Glycopegylated factor ix

ABSTRACT

The present invention provides conjugates between Factor IX and PEG moieties. The conjugates are linked via an intact glycosyl linking group interposed between and covalently attached to the peptide and the modifying group. The conjugates are formed from glycosylated peptides by the action of a glycosyltransferase. The glycosyltransferase ligates a modified sugar moiety onto a glycosyl residue on the peptide. Also provided are methods for preparing the conjugates, methods for treating various disease conditions with the conjugates, and pharmaceutical formulations including the conjugates.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/166,028, filed Jun. 23, 2005, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 60/684,729,filed May 25, 2005; U.S. patent application Ser. No. 11/166,028, filedJun. 23, 2005, which is a continuation-in-part of PCT Application No.PCT/US2004/041070, filed Dec. 3, 2004, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application No. 60/527,089,filed Dec. 3, 2003, U.S. Provisional Patent Application No. 60/539,387,filed Jan. 26, 2004, U.S. Provisional Patent Application No. 60/592,744,filed Jul. 29, 2004, U.S. Provisional Patent Application No. 60/614,518,filed Sep. 29, 2004, and U.S. Provisional Patent Application No.60/623,387, filed Oct. 29, 2004, each of which are incorporated hereinby reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Vitamin K-dependent proteins (e.g., Factor IX) contain 9 to 13gamma-carboxyglutamic acid residues (Gla) in their amino terminal 45residues. The Gla residues are produced by enzymes in the liver thatutilize vitamin K to carboxylate the side chains of glutamic acidresidues in protein precursors. Vitamin K-dependent proteins areinvolved in a number of biological processes, of which the bestdescribed is blood coagulation (reviewed in Nelsestuen, Vitam. Horm. 58:355-389 (2000)). Vitamin K-dependent proteins include protein Z, proteinS, prothrombin (Factor II), Factor X, Factor IX, protein C, Factor VII,Gas6, and matrix GLA protein. Factors VII, IX, X and II function inprocoagulation processes while protein C, protein S and protein Z servein anticoagulation roles. Gas6 is a growth arrest hormone encoded bygrowth arrest-specific gene 6 (gas6) and is related to protein S. See,Manfioletti et al. Mol. Cell. Biol. 13: 4976-4985 (1993). Matrix GLAprotein normally is found in bone and is critical to prevention ofcalcification of soft tissues in the circulation. Luo et al. Nature 386:78-81 (1997).

The regulation of blood coagulation is a process that presents a numberof leading health problems, including both the failure to form bloodclots as well as thrombosis, the formation of unwanted blood clots.Agents that prevent unwanted clots are used in many situations and avariety of agents are available. Unfortunately, most current therapieshave undesirable side effects. Orally administered anticoagulants suchas Warfarin act by inhibiting the action of vitamin K in the liver,thereby preventing complete carboxylation of glutamic acid residues inthe vitamin K-dependent proteins, resulting in a lowered concentrationof active proteins in the circulatory system and reduced ability to formclots. Warfarin therapy is complicated by the competitive nature of thedrug with its target. Fluctuations of dietary vitamin K can result in anover-dose or under-dose of Warfarin. Fluctuations in coagulationactivity are an undesirable outcome of this therapy.

Injected substances such as heparin, including low molecular weightheparin, also are commonly used anticoagulants. Again, these compoundsare subject to overdose and must be carefully monitored.

A newer category of anticoagulants includes active-site modified vitaminK-dependent clotting factors such as factor VIIa and IX^(a). The activesites are blocked by serine protease inhibitors such aschloromethylketone derivatives of amino acids or short peptides. Theactive site-modified proteins retain the ability to form complexes withtheir respective cofactors, but are inactive, thereby producing noenzyme activity and preventing complexing of the cofactor with therespective active enzymes. In short, these proteins appear to offer thebenefits of anticoagulation therapy without the adverse side effects ofother anticoagulants. Active site modified factor X^(a) is anotherpossible anticoagulant in this group. Its cofactor protein is factor Va.Active site modified activated protein C (APC) may also form aneffective inhibitor of coagulation. See, Sorensen et al. J. Biol. Chem.272: 11863-11868 (1997). Active site modified APC binds to factor Va andprevents factor X^(a) from binding.

A major inhibition to the use of vitamin K-dependent clotting factors iscost. Biosynthesis of vitamin K-dependent proteins is dependent on anintact glutamic acid carboxylation system, which is present in a smallnumber of animal cell types. Overproduction of these proteins is limitedby this enzyme system. Furthermore, the effective dose of these proteinsis high. A common dosage is 1000 μg of peptide/kg body weight. See,Harker et al. 1997, supra.

Another phenomena that hampers the use of therapeutic peptides is thewell known aspect of protein glycosylation is the relatively short invivo half life exhibited by these peptides. Overall, the problem of shotin vivo half life means that therapeutic glycopeptides must beadministered frequently in high dosages, which ultimately translate tohigher health care costs than might be necessary if a more efficientmethod for making longer lasting, more effective glycoproteintherapeutics was available.

Factor VIIa, for example, illustrates this problem. Factor VII and VIIahave circulation half-times of about 2-4 hours in the human. That is,within 2-4 hours, the concentration of the peptide in the serum isreduced by half. When Factor VIIa is used as a procoagulant to treatcertain forms of hemophilia, the standard protocol is to inject VIIaevery two hours and at high dosages (45 to 90 .mu.g/kg body weight).See, Hedner et al., Transfus. Med. Rev. 7: 78-83 (1993)). Thus, use ofthese proteins as procoagulants or anticoagulants (in the case of factorVIIa) requires that the proteins be administered at frequent intervalsand at high dosages.

One solution to the problem of providing cost effective glycopeptidetherapeutics has been to provide peptides with longer in vivo halflives. For example, glycopeptide therapeutics with improvedpharmacokinetic properties have been produced by attaching syntheticpolymers to the peptide backbone. An exemplary polymer that has beenconjugated to peptides is poly(ethylene glycol) (“PEG”). The use of PEGto derivatize peptide therapeutics has been demonstrated to reduce theimmunogenicity of the peptides. For example, U.S. Pat. No. 4,179,337(Davis et al.) discloses non-immunogenic polypeptides such as enzymesand peptide hormones coupled to polyethylene glycol (PEG) orpolypropylene glycol. In addition to reduced immunogenicity, theclearance time in circulation is prolonged due to the increased size ofthe PEG-conjugate of the polypeptides in question.

The principal mode of attachment of PEG, and its derivatives, topeptides is a non-specific bonding through a peptide amino acid residue(see e.g., U.S. Pat. No. 4,088,538 U.S. Pat. No. 4,496,689, U.S. Pat.No. 4,414,147, U.S. Pat. No. 4,055,635, and PCT WO 87/00056). Anothermode of attaching PEG to peptides is through the non-specific oxidationof glycosyl residues on a glycopeptide (see e.g., WO 94/05332).

In these non-specific methods, poly(ethyleneglycol) is added in arandom, non-specific manner to reactive residues on a peptide backbone.Of course, random addition of PEG molecules has its drawbacks, includinga lack of homogeneity of the final product, and the possibility forreduction in the biological or enzymatic activity of the peptide.Therefore, for the production of therapeutic peptides, a derivitizationstrategy that results in the formation of a specifically labeled,readily characterizable, essentially homogeneous product is superior.Such methods have been developed.

Specifically labeled, homogeneous peptide therapeutics can be producedin vitro through the action of enzymes. Unlike the typical non-specificmethods for attaching a synthetic polymer or other label to a peptide,enzyme-based syntheses have the advantages of regioselectivity andstereoselectivity. Two principal classes of enzymes for use in thesynthesis of labeled peptides are glycosyltransferases (e.g.,sialyltransferases, oligosaccharyltransferases,N-acetylglucosaminyltransferases), and glycosidases. These enzymes canbe used for the specific attachment of sugars which can be subsequentlymodified to comprise a therapeutic moiety. Alternatively,glycosyltransferases and modified glycosidases can be used to directlytransfer modified sugars to a peptide backbone (see e.g., U.S. Pat. No.6,399,336, and U.S. Patent Application Publications 20030040037,20040132640, 20040137557, 20040126838, and 20040142856, each of whichare incorporated by reference herein). Methods combining both chemicaland enzymatic synthetic elements are also known (see e.g., Yamamoto etal. Carbohydr. Res. 305: 415-422 (1998) and U.S. Patent ApplicationPublication 20040137557 which is incorporated herein by reference).

Factor IX is an extremely valuable therapeutic peptide. Althoughcommercially available forms of Factor IX are in use today, thesepeptides can be improved by modifications that enhance thepharmacokinetics of the resulting isolated glycoprotein product. Thus,there remains a need in the art for longer lasting Factor IX peptideswith improved effectiveness and better pharmacokinetics. Furthermore, tobe effective for the largest number of individuals, it must be possibleto produce, on an industrial scale, a Factor IX peptide with improvedtherapeutic pharmacokinetics that has a predictable, essentiallyhomogeneous, structure which can be readily reproduced over, and overagain.

Fortunately, Factor IX peptides with improved pharmacokinetics andmethods for making them have now been discovered. In addition to FactorIX peptides with improved pharmacokinetics, the invention also providesindustrially practical and cost effective methods for the production ofthese Factor IX peptides. The Factor IX peptides of the inventioncomprise modifying groups such as PEG moieties, therapeutic moieties,biomolecules and the like. The present invention therefore fulfills theneed for Factor IX peptides with improved the therapeutic effectivenessand improved pharmacokinetics for the treatment of conditions anddiseases wherein Factor IX provides effective therapy.

SUMMARY OF THE INVENTION

It has now been discovered that the controlled modification of Factor IXwith one or more poly(ethylene glycol) moieties affords a novel FactorIX derivative with pharmacokinetic properties that are improved relativeto the corresponding native (un-pegylated) Factor IX (FIG. 3). Moreover,the glycoPEGylated Factor IX retains its pharmacological activity (FIG.4).

In an exemplary embodiment, “glycopeglyated” Factor IX molecules of theinvention are produced by the enzyme mediated formation of a conjugatebetween a glycosylated or non-glycosylated Factor IX peptide and anenzymatically transferable saccharyl moiety that includes apoly(ethylene glycol) moiety within its structure The PEG moiety isattached to the saccharyl moiety directly (i.e., through a single groupformed by the reaction of two reactive groups) or through a linkermoiety, e.g., substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, etc. An exemplary transferable PEG-saccharylstructure is set forth in FIG. 7.

The polymeric modifying moiety can be attached at any position of aglycosyl moiety of Factor IX. Moreover, the polymeric modifying moietycan be bound to a glycosyl residue at any position in the amino acidsequence of a wild type or mutant Factor IX peptide.

In an exemplary embodiment, the invention provides an Factor IX peptidethat is conjugated through a glycosyl linking group to a polymericmodifying moiety. Exemplary Factor IX peptide conjugates include aglycosyl linking group having a formula selected from:

In Formulae I and II, R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷represents H, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl. The symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl or sialic acid. At least one of R³, R⁴, R⁵, R⁶ or R^(6′)includes the polymeric modifying moiety e.g., PEG. In an exemplaryembodiment, R⁶ and R^(6′), together with the carbon to which they areattached are components of the side chain of sialic acid. In a furtherexemplary embodiment, this side chain is functionalized with thepolymeric modifying moiety.

In an exemplary embodiment, the polymeric moiety is bound to theglycosyl linking group, generally through a heteroatom on the glycosylcore (e.g., N, O), through a linker, L, as shown below:

R¹ is the polymeric modifying moiety and L is selected from a bond and alinking group. The index w represents an integer selected from 1-6,preferably 1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety. Another exemplary linking group is an aminoacid residue (e.g., cysteine, serine, lysine, and short oligopeptides,e.g., Lys-Lys, Lys-Lys-Lys, Cys-Lys, Ser-Lys, etc.)

When L is a bond, it is formed by reaction of a reactive functionalgroup on a precursor of R¹ and a reactive functional group ofcomplementary reactivity on a precursor of the glycosyl linking group.When L is a non-zero order linking group, L can be in place on theglycosyl moiety prior to reaction with the R¹ precursor. Alternatively,the precursors of R¹ and L can be incorporated into a preformed cassettethat is subsequently attached to the glycosyl moiety. As set forthherein, the selection and preparation of precursors with appropriatereactive functional groups is within the ability of those skilled in theart. Moreover, coupling of the precursors proceeds by chemistry that iswell understood in the art.

In an exemplary embodiment L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. Amino acid analogs, as defined herein, arealso of use as linker components. The amino acid may be modified with anadditional component of a linker, e.g., alkyl, heteroalkyl, covalentlyattached through an acyl linkage, for example, an amide or urethaneformed through an amine moiety of the amino acid residue.

In an exemplary embodiment, the glycosyl linker has a structureaccording to Formula I and R⁵ includes the polymeric modifying moiety.In another exemplary embodiment, R⁵ includes both the polymericmodifying moiety and a linker, L, joining the modifying moiety to theglycosyl core. L can be a linear or branched structure. Similarly, thepolymeric modifying can be branched or linear.

The polymeric modifying moiety comprises two or more repeating unitsthat can be water-soluble or essentially insoluble in water. Exemplarywater-soluble polymers of use in the compounds of the invention includePEG, e.g., m-PEG, PPG, e.g., m-PPG, polysialic acid, polyglutamate,polyaspartate, polylysine, polyethyeleneimine, biodegradable polymers(e.g., polylactide, polyglyceride), and functionalized PEG, e.g.,terminal-functionalized PEG.

The glycosyl core of the glycosyl linking groups of use in the Factor IXconjugates of the invention is selected from both natural and unnaturalfuranoses and pyranoses. The unnatural saccharides optionally include analkylated or acylated hydroxyl and/or amine moiety, e.g., ethers, estersand amide substituents on the ring. Other unnatural saccharides includean H, hydroxyl, ether, ester or amide substituent at a position on thering at which such a substituent is not present in the naturalsaccharide. Alternatively, the carbohydrate is missing a substituentthat would be found in the carbohydrate from which its name is derived,e.g., deoxy sugars. Still further exemplary unnatural sugars includeboth oxidized (e.g., -onic and -uronic acids) and reduced (sugaralcohols) carbohydrates. The sugar moiety can be a mono-, oligo- orpoly-saccharide.

Exemplary natural sugars of use as components of glycosyl linking groupsin the present invention include glucose, glucosamine, galactose,galactosamine, fucose, mannose, mannosamine, xylanose, ribose, N-acetylglucose, N-acetyl glucosamine, N-acetyl galactose, N-acetylgalactosamine, and sialic acid.

In one embodiment, the present invention provides an Factor IX peptidecomprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another aspect, the invention provides a peptide comprising aglycosyl linking group having the formula:

In other embodiments, the group has the formula:

in which t is 0 or 1.

In yet another embodiment, the group has the formula:

in which the index p represents and integer from 1 to 10, and crepresents 0 or 1.

In another aspect, the invention provides a method of making a PEGylatedFactor IX peptide of the invention. The method includes: (a) contactinga substrate Factor IX peptide comprising a glycosyl group selected from:

with a PEG-sialic acid donor having the formula:

and an enzyme that transfers PEG-sialic acid from said donor onto amember selected from the GalNAc, Gal and the Sia of said glycosyl group,under conditions appropriate for said transfer. An exemplary modifiedsialic acid donor is CMP-sialic acid modified, through a linker moiety,with a polymer, e.g., a straight chain or branched poly(ethylene glycol)moiety. The indices c and r independently represent 0 or 1.

The peptide can be acquired from essentially any source, however, in oneembodiment, prior to being modified as discussed above, the Factor IXpeptide is expressed in a suitable host. Mammalian (e.g., CHO), bacteria(e.g., E. coli) and insect cells (e.g., Sf-9) are exemplary expressionsystems providing Factor IX of use in the compositions and methods setforth herein. An exemplary O-linked glycan that is glycopegylated isshown in FIG. 9. Exemplary glycans produced in an insect system and amammalian system, and subsequently glycoconjugated and or remodeled andglycoconjugated to PEG are set forth in FIG. 10 and FIG. 11.

In another aspect, the invention provides a method of treating acondition in a subject in need thereof. Exemplary conditions includethose characterized by compromised blood clotting in the subject. Themethod includes the step of administering to the subject an amount ofthe polymer-modified Factor IX peptide of the invention effective toameliorate the condition in the subject.

In another aspect, the invention provides a method of enhancing bloodclotting in a mammal. The method includes administering to the mammal anamount of the polymer-modified Factor IX peptide of the inventioneffective to enhance clotting in the mammal.

In another aspect, the invention provides a method of treating acondition in a subject in need of treatment with Factor IX. The methodincludes the step of administering to the subject an amount of apolymer-modified Factor IX peptide of the invention effective toameliorate the condition of the subject.

In another aspect, the invention provides a pharmaceutical formulationcomprising a polymer-modified Factor IX peptide of the invention and apharmaceutically acceptable carrier.

In the polymer-modified Factor IX glycoconjugates of the invention,essentially each of the amino acid residues to which the polymer isbound has the same structure. For example, if one peptide includes anasparagine linked glycosyl residue, at least about 70%, 80%, 90%, 95%,97%, 99%, 99.2%, 99.4%, 99.6%, or more preferably 99.8% of the peptidesin the population will have the same glycosyl residue covalently boundto the same Ser residue. In other embodiments, this is true of aglycosyl residue linked to a threonine or a serine.

Other objects and advantages of the invention will be apparent to thoseof skill in the art from the detailed description that follows.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of Factor IX, showing the presence and locationof potential glycosylation sites at Asn 157, Asn 167; Ser 53, Ser 61,Thr 159, Thr 169, and Thr 172.

FIG. 2 is a scheme showing an exemplary embodiment of the invention inwhich a carbohydrate residue on a Factor IX peptide is remodeled andglycopegylated: (A) sialic acid moieties are removed by sialidase andthe resulting galactose residues are glycopegylated with the sialic acidderivative of FIG. 5; (B) a mannose residue is glycopegylated with thesialic acid PEG; (C) a sialic acid moiety of an N-glycan isglycopegylated with the sialic acid PEG (a′ and a″ are independentlyselected from 0 and 1-at least one of a′ and a″ is 1); The figure isexemplary in that any glycosylated Factor IX molecule may comprise anymixture of mono-, bi- tri-, or tetra-antennary N-linked glycosylresidues and any one or more of the branches may further comprise amodified sialic acid moiety of the invention. Moreover, the figureillustrates that the modified glycan can be positioned at any one ormore N- or O-linked glycosylation site without limitation.

FIG. 3 is a plot comparing the in vivo residence lifetimes ofunglycosylated Factor IX and enzymatically glycoPEGylated Factor IX.

FIG. 4 is a table comparing the activities of the species shown in FIG.3.

FIG. 5 is the amino acid sequence of Factor IX.

FIG. 6 is a graphic presentation of the pharmacokinetic properties ofvarious glycoPEGylated Factor IX molecules compared to a non-pegylatedFactor IX. LS refers to “low substitution” (the peptide isglycoPEGylated using ST3Gal3 without desialylation). HS refers to highsubstitution (the peptide is glycoPEGylated using ST3Gal3, followingdesialylation). Unmodified Gal residues are optionally capped with Sia.

FIG. 7 is a synthetic scheme for producing an exemplary PEG-glycosyllinking group precursor (modified sugar) of us in preparing theconjugates of the invention.

FIG. 8 is a table of sialyl transferases of use to transfer onto anacceptor a modified sialic acid moiety, such as those set forth hereinand unmodified sialic acid moieties.

FIG. 9 shows an exemplary O-linked glycan structure on a Factor IXglycoconjugate of the invention. Each index n is independently selected.

FIG. 10 shows an exemplary N-linked glycan structure on a mutant FactorIX glycoconjugate of the invention expressed in insect cells (andremodeled and glycopegylated) in which the mutant includes one or moreN-linked glycosylation sites.

FIG. 11 shows an exemplary N-linked glycan structure on a mutant FactorIX glycoconjugate of the invention expressed in mammalian cells (andglycopegylated) in which the mutant includes one or more N-linkedglycosylation sites: A) N-linked glycans of Factor IX expressed in CHO;B) N-linked glycans of CHO-derived Factor IX glycoPEGylated with CST-IIor α2,8 sialyltransferase; C)N-linked glycans of CHO-derived Factor IXglycoPEGylated with CST-II and/or ST3Gal3. A glycine linker can beinterposed between the linear and/or branched PEG species such asdiscussed herein.

FIG. 12 illustrates exemplary modified sialic acid nucleotides useful inthe practice of the invention. A. Structure of exemplary branched (e.g.,30 kDa, 40 kDa) CMP-sialic acid-PEG sugar nucleotides. B. Structure oflinear CMP-sialic acid-PEG (e.g., 10 kDa).

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTSAbbreviations

PEG, poly(ethylene glycol); PPG, poly(propylene glycol); Ara,arabinosyl; Fru, fructosyl; Fuc, fucosyl; Gal, galactosyl; GalNAc,N-acetylgalactosaminyl; Glc, glucosyl; GlcNAc, N-acetylglucosaminyl;Man, mannosyl; ManAc, mannosaminyl acetate; Xyl, xylosyl; NeuAc(N-acetylneuraminyl), Sia (sialyl); M6P, mannose-6-phosphate.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization are those well known and commonly employedin the art. Standard techniques are used for nucleic acid and peptidesynthesis. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

All oligosaccharides described herein are described with the name orabbreviation for the non-reducing saccharide (i.e., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond (1 or2), the ring position of the reducing saccharide involved in the bond(2, 3, 4, 6 or 8), and then the name or abbreviation of the reducingsaccharide (i.e., GlcNAc). Each saccharide is preferably a pyranose. Fora review of standard glycobiology nomenclature, see, Essentials ofGlycobiology Varki et al. eds. CSHL Press (1999).

Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetyl-neuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem. 265:21811-21819 (1990)). Also included are 9-substituted sialic acids suchas a 9-O—C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki, Glycobiology 2: 25-40 (1992);Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992)). The synthesis and use of sialic acidcompounds in a sialylation procedure is disclosed in internationalapplication WO 92/16640, published Oct. 1, 1992.

“Peptide” refers to a polymer in which the monomers are amino acids andare joined together through amide bonds, alternatively referred to as apolypeptide. Additionally, unnatural amino acids, for example,β-alanine, phenylglycine and homoarginine are also included. Amino acidsthat are not gene-encoded may also be used in the present invention.Furthermore, amino acids that have been modified to include reactivegroups, glycosylation sites, polymers, therapeutic moieties,biomolecules and the like may also be used in the invention. All of theamino acids used in the present invention may be either the D- orL-isomer. The L-isomer is generally preferred. In addition, otherpeptidomimetics are also useful in the present invention. As usedherein, “peptide” refers to both glycosylated and unglycosylatedpeptides. Also included are peptides that are incompletely glycosylatedby a system that expresses the peptide. For a general review, see,Spatola, A. F., in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDESAND PROTEINS, B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983).

The term “peptide conjugate,” refers to species of the invention inwhich a peptide is conjugated with a modified sugar as set forth herein.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

As used herein, the term “modified sugar,” refers to a naturally- ornon-naturally-occurring carbohydrate that is enzymatically added onto anamino acid or a glycosyl residue of a peptide in a process of theinvention. The modified sugar is selected from enzyme substratesincluding, but not limited to sugar nucleotides (mono-, di-, andtri-phosphates), activated sugars (e.g., glycosyl halides, glycosylmesylates) and sugars that are neither activated nor nucleotides. The“modified sugar” is covalently functionalized with a “modifying group.”Useful modifying groups include, but are not limited to, PEG moieties,therapeutic moieties, diagnostic moieties, biomolecules and the like.The modifying group is preferably not a naturally occurring, or anunmodified carbohydrate. The locus of functionalization with themodifying group is selected such that it does not prevent the “modifiedsugar” from being added enzymatically to a peptide.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like. Peptides can have mixed sequencesof be composed of a single amino acid, e.g., poly(lysine). An exemplarypolysaccharide is poly(sialic acid). An exemplary poly(ether) ispoly(ethylene glycol). Poly(ethylene imine) is an exemplary polyamine,and poly(acrylic) acid is a representative poly(carboxylic acid).

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (i.e. PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(-PEG-OH)_(m) in which R represents the core moiety,such as glycerol or pentaerythritol, and m represents the number ofarms. Multi-armed PEG molecules, such as those described in U.S. Pat.No. 5,932,462, which is incorporated by reference herein in itsentirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxypropylmethacrylamide), poly(α-hydroxy acid), poly(vinylalcohol), polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine),such as described in U.S. Pat. No. 5,629,384, which is incorporated byreference herein in its entirety, and copolymers, terpolymers, andmixtures thereof. Although the molecular weight of each chain of thepolymer backbone can vary, it is typically in the range of from about100 Da to about 100,000 Da, often from about 6,000 Da to about 80,000Da.

The “area under the curve” or “AUC”, as used herein in the context ofadministering a peptide drug to a patient, is defined as total areaunder the curve that describes the concentration of drug in systemiccirculation in the patient as a function of time from zero to infinity.

The term “half-life” or “t½”, as used herein in the context ofadministering a peptide drug to a patient, is defined as the timerequired for plasma concentration of a drug in a patient to be reducedby one half. There may be more than one half-life associated with thepeptide drug depending on multiple clearance mechanisms, redistribution,and other mechanisms well known in the art. Usually, alpha and betahalf-lives are defined such that the alpha phase is associated withredistribution, and the beta phase is associated with clearance.However, with protein drugs that are, for the most part, confined to thebloodstream, there can be at least two clearance half-lives. For someglycosylated peptides, rapid beta phase clearance may be mediated viareceptors on macrophages, or endothelial cells that recognize terminalgalactose, N-acetylgalactosamine, N-acetylglucosamine, mannose, orfucose. Slower beta phase clearance may occur via renal glomerularfiltration for molecules with an effective radius <2 nm (approximately68 kD) and/or specific or non-specific uptake and metabolism in tissues.GlycoPEGylation may cap terminal sugars (e.g., galactose orN-acetylgalactosamine) and thereby block rapid alpha phase clearance viareceptors that recognize these sugars. It may also confer a largereffective radius and thereby decrease the volume of distribution andtissue uptake, thereby prolonging the late beta phase. Thus, the preciseimpact of glycoPEGylation on alpha phase and beta phase half-lives willvary depending upon the size, state of glycosylation, and otherparameters, as is well known in the art. Further explanation of“half-life” is found in Pharmaceutical Biotechnology (1997, DFACrommelin and RD Sindelar, eds., Harwood Publishers, Amsterdam, pp101-120).

The term “glycoconjugation,” as used herein, refers to the enzymaticallymediated conjugation of a modified sugar species to an amino acid orglycosyl residue of a polypeptide, e.g., a Factor IX peptide of thepresent invention. A subgenus of “glycoconjugation” is“glycol-PEGylation,” in which the modifying group of the modified sugaris poly(ethylene glycol), and alkyl derivative (e.g., m-PEG) or reactivederivative (e.g., H₂N-PEG, HOOC-PEG) thereof.

The terms “large-scale” and “industrial-scale” are used interchangeablyand refer to a reaction cycle that produces at least about 250 mg,preferably at least about 500 mg, and more preferably at least about 1gram of glycoconjugate at the completion of a single reaction cycle.

The term, “glycosyl linking group,” as used herein refers to a glycosylresidue to which a modifying group (e.g., PEG moiety, therapeuticmoiety, biomolecule) is covalently attached; the glycosyl linking groupjoins the modifying group to the remainder of the conjugate. In themethods of the invention, the “glycosyl linking group” becomescovalently attached to a glycosylated or unglycosylated peptide, therebylinking the agent to an amino acid and/or glycosyl residue on thepeptide. A “glycosyl linking group” is generally derived from a“modified sugar” by the enzymatic attachment of the “modified sugar” toan amino acid and/or glycosyl residue of the peptide. The glycosyllinking group can be a saccharide-derived structure that is degradedduring formation of modifying group-modified sugar cassette (e.g.,oxidation→Schiff base formation→reduction), or the glycosyl linkinggroup may be intact. An “intact glycosyl linking group” refers to alinking group that is derived from a glycosyl moiety in which thesaccharide monomer that links the modifying group and to the remainderof the conjugate is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate. “Intact glycosyl linking groups” of the invention may bederived from a naturally occurring oligosaccharide by addition ofglycosyl unit(s) or removal of one or more glycosyl unit from a parentsaccharide structure.

The term “targeting moiety,” as used herein, refers to species that willselectively localize in a particular tissue or region of the body. Thelocalization is mediated by specific recognition of moleculardeterminants, molecular size of the targeting agent or conjugate, ionicinteractions, hydrophobic interactions and the like. Other mechanisms oftargeting an agent to a particular tissue or region are known to thoseof skill in the art. Exemplary targeting moieties include antibodies,antibody fragments, transferrin, HS-glycoprotein, coagulation factors,serum proteins, β-glycoprotein, G-CSF, GM-CSF, M-CSF, EPO and the like.

As used herein, “therapeutic moiety” means any agent useful for therapyincluding, but not limited to, antibiotics, anti-inflammatory agents,anti-tumor drugs, cytotoxins, and radioactive agents. “Therapeuticmoiety” includes prodrugs of bioactive agents, constructs in which morethan one therapeutic moiety is bound to a carrier, e.g, multivalentagents. Therapeutic moiety also includes proteins and constructs thatinclude proteins. Exemplary proteins include, but are not limited to,Granulocyte Colony Stimulating Factor (GCSF), Granulocyte MacrophageColony Stimulating Factor (GMCSF), Interferon (e.g., Interferon-α, -β,-γ), Interleukin (e.g., Interleukin II), serum proteins (e.g., FactorsVII, VIIa, VIII, IX, and X), Human Chorionic Gonadotropin (HCG),Follicle Stimulating Hormone (FSH) and Lutenizing Hormone (LH) andantibody fusion proteins (e.g. Tumor Necrosis Factor Receptor ((TNFR)/Fcdomain fusion protein)).

As used herein, “pharmaceutically acceptable carrier” includes anymaterial, which when combined with the conjugate retains the conjugates'activity and is non-reactive with the subject's immune systems. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, emulsionssuch as oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets including coatedtablets and capsules. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gums, glycols, or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well known conventionalmethods.

As used herein, “administering,” means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular, intralesional, intranasal orsubcutaneous administration, or the implantation of a slow-releasedevice e.g., a mini-osmotic pump, to the subject. Administration is byany route including parenteral, and transmucosal (e.g., oral, nasal,vaginal, rectal, or transdermal). Parenteral administration includes,e.g., intravenous, intramuscular, intra-arteriole, intradermal,subcutaneous, intraperitoneal, intraventricular, and intracranial.Moreover, where injection is to treat a tumor, e.g., induce apoptosis,administration may be directly to the tumor and/or into tissuessurrounding the tumor. Other modes of delivery include, but are notlimited to, the use of liposomal formulations, intravenous infusion,transdermal patches, etc.

The term “ameliorating” or “ameliorate” refers to any indicia of successin the treatment of a pathology or condition, including any objective orsubjective parameter such as abatement, remission or diminishing ofsymptoms or an improvement in a patient's physical or mental well-being.Amelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination and/or apsychiatric evaluation.

The term “therapy” refers to “treating” or “treatment” of a disease orcondition including preventing the disease or condition from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease).

The term “effective amount” or “an amount effective to” or a“therapeutically effective amount” or any grammatically equivalent termmeans the amount that, when administered to an animal for treating adisease, is sufficient to effect treatment for that disease.

The term “isolated” refers to a material that is substantially oressentially free from components, which are used to produce thematerial. For peptide conjugates of the invention, the term “isolated”refers to material that is substantially or essentially free fromcomponents which normally accompany the material in the mixture used toprepare the peptide conjugate. “Isolated” and “pure” are usedinterchangeably. Typically, isolated peptide conjugates of the inventionhave a level of purity preferably expressed as a range. The lower end ofthe range of purity for the peptide conjugates is about 60%, about 70%or about 80% and the upper end of the range of purity is about 70%,about 80%, about 90% or more than about 90%.

When the peptide conjugates are more than about 90% pure, their puritiesare also preferably expressed as a range. The lower end of the range ofpurity is about 90%, about 92%, about 94%, about 96% or about 98%. Theupper end of the range of purity is about 92%, about 94%, about 96%,about 98% or about 100% purity.

Purity is determined by any art-recognized method of analysis (e.g.,band intensity on a silver stained gel, polyacrylamide gelelectrophoresis, HPLC, or a similar means).

“Essentially each member of the population,” as used herein, describes acharacteristic of a population of peptide conjugates of the invention inwhich a selected percentage of the modified sugars added to a peptideare added to multiple, identical acceptor sites on the peptide.“Essentially each member of the population” speaks to the “homogeneity”of the sites on the peptide conjugated to a modified sugar and refers toconjugates of the invention, which are at least about 80%, preferably atleast about 90% and more preferably at least about 95% homogenous.

“Homogeneity,” refers to the structural consistency across a populationof acceptor moieties to which the modified sugars are conjugated. Thus,in a peptide conjugate of the invention in which each modified sugarmoiety is conjugated to an acceptor site having the same structure asthe acceptor site to which every other modified sugar is conjugated, thepeptide conjugate is said to be about 100% homogeneous. Homogeneity istypically expressed as a range. The lower end of the range ofhomogeneity for the peptide conjugates is about 60%, about 70% or about80% and the upper end of the range of purity is about 70%, about 80%,about 90% or more than about 90%.

When the peptide conjugates are more than or equal to about 90%homogeneous, their homogeneity is also preferably expressed as a range.The lower end of the range of homogeneity is about 90%, about 92%, about94%, about 96% or about 98%. The upper end of the range of purity isabout 92%, about 94%, about 96%, about 98% or about 100% homogeneity.The purity of the peptide conjugates is typically determined by one ormore methods known to those of skill in the art, e.g., liquidchromatography-mass spectrometry (LC-MS), matrix assisted laserdesorption mass time of flight spectrometry (MALDITOF), capillaryelectrophoresis, and the like.

“Substantially uniform glycoform” or a “substantially uniformglycosylation pattern,” when referring to a glycopeptide species, refersto the percentage of acceptor moieties that are glycosylated by theglycosyltransferase of interest (e.g., fucosyltransferase). For example,in the case of a α1,2 fucosyltransferase, a substantially uniformfucosylation pattern exists if substantially all (as defined below) ofthe Galβ1,4-GlcNAc-R and sialylated analogues thereof are fucosylated ina peptide conjugate of the invention. It will be understood by one ofskill in the art, that the starting material may contain glycosylatedacceptor moieties (e.g., fucosylated Galβ1,4-GlcNAc-R moieties). Thus,the calculated percent glycosylation will include acceptor moieties thatare glycosylated by the methods of the invention, as well as thoseacceptor moieties already glycosylated in the starting material.

The term “substantially” in the above definitions of “substantiallyuniform” generally means at least about 40%, at least about 70%, atleast about 80%, or more preferably at least about 90%, and still morepreferably at least about 95% of the acceptor moieties for a particularglycosyltransferase are glycosylated.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂-S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, substituent that can be a single ring or multiple rings(preferably from 1 to 3 rings), which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl and heteroaryl groups include phenyl, 1-naphthyl,2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl,2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl and 6-quinolyl.Substituents for each of the above noted aryl and heteroaryl ringsystems are selected from the group of acceptable substituents describedbelow.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)=NR″″,—NR—C(NR′R″)=NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)-U-, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

Introduction

As described above, Factor IX is vital in the blood coagulation cascade.The structure and sequence of Factor IX is provided in FIG. 1 and FIG.5. A deficiency of Factor IX in the body characterizes a type ofhemophilia (type B). Treatment of this disease is usually limited tointravenous transfusion of human plasma protein concentrates of FactorIX. However, in addition to the practical disadvantages of time andexpense, transfusion of blood concentrates involves the risk oftransmission of viral hepatitis, acquired immune deficiency syndrome orthromboembolic diseases to the recipient.

While Factor IX is an important and useful compound for therapeuticapplications, present methods for the production of Factor IX fromrecombinant cells (U.S. Pat. No. 4,770,999) result in a product with arather short biological half-life and an inaccurate glycosylationpattern that could potentially lead to immunogenicity, loss of function,an increased need for both larger and more frequent doses in order toachieve the same effect, and the like.

To improve the effectiveness of recombinant Factor IX used fortherapeutic purposes, the present invention provides conjugates ofglycosylated and unglycosylated Factor IX peptides with polymers, e.g.,PEG (m-PEG), PPG (m-PPG), etc. The conjugates may be additionally oralternatively modified by further conjugation with diverse species suchas therapeutic moieties, diagnostic moieties, targeting moieties and thelike.

The conjugates of the invention are formed by the enzymatic attachmentof a modified sugar to the glycosylated or unglycosylated peptide. Aglycosylation site and/or a glycosyl residue provides a locus forconjugating a sugas bearing a modifying group to the peptide, e.g., byglycoconjugation. An exemplary modifying group is a water-solublepolymer, such as poly(ethylene glycol), e.g., methoxy-poly(ethyleneglycol). Modification of the Factor IX peptides, e.g., with awater-soluble peptide can improve the stability and retention time ofthe recombinant Factor IX in a patient's circulation, and/or reduce theantigenicity of recombinant Factor IX.

The methods of the invention make it possible to assemble peptides andglycopeptides that have a substantially homogeneous derivatizationpattern. The enzymes used in the invention are generally selective for aparticular amino acid residue, combination of amino acid residues, orparticular glycosyl residues of the peptide. The methods are alsopractical for large-scale production of modified peptides andglycopeptides. Thus, the methods of the invention provide a practicalmeans for large-scale preparation of glycopeptides having preselecteduniform derivatization patterns.

The present invention also provides conjugates of glycosylated andunglycosylated peptides with increased therapeutic half-life due to, forexample, reduced clearance rate, or reduced rate of uptake by the immuneor reticuloendothelial system (RES). Moreover, the methods of theinvention provide a means for masking antigenic determinants onpeptides, thus reducing or eliminating a host immune response againstthe peptide. Selective attachment of targeting agents can also be usedto target a peptide to a particular tissue or cell surface receptor thatis specific for the particular targeting agent.

The Conjugates

In a first aspect, the present invention provides a conjugate between aselected modifying group and an Factor IX peptide.

The link between the peptide and the modifying moiety includes aglycosyl linking group interposed between the peptide and the selectedmoiety. As discussed herein, the selected modifying moiety isessentially any species that can be attached to a saccharide unit,resulting in a “modified sugar” that is recognized by an appropriatetransferase enzyme, which appends the modified sugar onto the peptide,or a glycosyl residue attached thereto. The saccharide component of themodified sugar, when interposed between the peptide and a selectedmoiety, becomes a “glycosyl linking group,” e.g., an “intact glycosyllinking group.” The glycosyl linking group is formed from any mono- oroligo-saccharide that, after modification with the modifying group, is asubstrate for an enzyme that adds the modified sugar to an amino acid orglycosyl residue of a peptide.

The glycosyl linking group can be, or can include, a saccharide moietythat is degradatively modified before or during the addition of themodifying group. For example, the glycosyl linking group can be derivedfrom a saccharide residue that is produced by oxidative degradation ofan intact saccharide to the corresponding aldehyde, e.g., via the actionof metaperiodate, and subsequently converted to a Schiff base with anappropriate amine, which is then reduced to the corresponding amine.

The conjugates of the invention will typically correspond to the generalstructure:

in which the symbols a, b, c, d and s represent a positive, non-zerointeger; and t is either 0 or a positive integer. The “agent” is atherapeutic agent, a bioactive agent, a detectable label, water-solublemoiety (e.g., PEG, m-PEG, PPG, and m-PPG) or the like. The “agent” canbe a peptide, e.g., enzyme, antibody, antigen, etc. The linker can beany of a wide array of linking groups, infra. Alternatively, the linkermay be a single bond or a “zero order linker.”

In an exemplary embodiment, the selected modifying group is awater-soluble polymer, e.g., m-PEG. The water-soluble polymer iscovalently attached to the peptide via a glycosyl linking group. Theglycosyl linking group is covalently attached to an amino acid residueor a glycosyl residue of the peptide. The invention also providesconjugates in which an amino acid residue and a glycosyl residue aremodified with a glycosyl linking group.

An exemplary water-soluble polymer is poly(ethylene glycol), e.g.,methoxy-poly(ethylene glycol). The poly(ethylene glycol) used in thepresent invention is not restricted to any particular form or molecularweight range. For unbranched poly(ethylene glycol) molecules themolecular weight is preferably between 500 and 100,000. A molecularweight of 2000-60,000 is preferably used and preferably of from about5,000 to about 30,000.

In another embodiment the poly(ethylene glycol) is a branched PEG havingmore than one PEG moiety attached. Examples of branched PEGs aredescribed in U.S. Pat. No. 5,932,462; U.S. Pat. No. 5,342,940; U.S. Pat.No. 5,643,575; U.S. Pat. No. 5,919,455; U.S. Pat. No. 6,113,906; U.S.Pat. No. 5,183,660; WO 02/09766; Kodera Y., Bioconjugate Chemistry 5:283-288 (1994); and Yamasaki et al., Agric. Biol. Chem., 52: 2125-2127,1998. In a preferred embodiment the molecular weight of eachpoly(ethylene glycol) of the branched PEG is less than or equal to40,000 daltons.

In addition to providing conjugates that are formed through anenzymatically added glycosyl linking group, the present inventionprovides conjugates that are highly homogenous in their substitutionpatterns. Using the methods of the invention, it is possible to formpeptide conjugates in which essentially all of the modified sugarmoieties across a population of conjugates of the invention are attachedto a structurally identical amino acid or glycosyl residue. Thus, in asecond aspect, the invention provides a peptide conjugate having apopulation of water-soluble polymer moieties, which are covalently boundto the peptide through a glycosyl linking group, e.g., an intactglycosyl linking group. In a preferred conjugate of the invention,essentially each member of the population is bound via the glycosyllinking group to a glycosyl residue of the peptide, and each glycosylresidue of the peptide to which the glycosyl linking group is attachedhas the same structure.

Also provided is a peptide conjugate having a population ofwater-soluble polymer moieties covalently bound thereto through aglycosyl linking group. In a preferred embodiment, essentially everymember of the population of water soluble polymer moieties is bound toan amino acid residue of the peptide via a glycosyl linking group, andeach amino acid residue having a glycosyl linking group attached theretohas the same structure.

The present invention also provides conjugates analogous to thosedescribed above in which the peptide is conjugated to a therapeuticmoiety, diagnostic moiety, targeting moiety, toxin moiety or the likevia an intact glycosyl linking group. Each of the above-recited moietiescan be a small molecule, natural polymer (e.g., polypeptide) orsynthetic polymer. When the modifying moiety is attached to a sialicacid, it is generally preferred that the modifying moiety issubstantially non-fluorescent.

In an exemplary embodiment, in which the glycosyl residue has thestructure set forth above, it is conjugated to one or both Asn 157 andAsn 167.

Factor IX has been cloned and sequenced. Essentially any Factor IXpeptide having any sequence is of use as the Factor IX peptide componentof the conjugates of the present invention. In an exemplary embodiment,the peptide has the sequence presented herein as SEQ ID NO:1:

YNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAEAVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTK VSRYVNWIKEKTKLT.

The present invention is in no way limited to the sequence set forthherein. Factor IX variants are well known in the art, as described in,for example, U.S. Pat. Nos. 4,770,999, 5,521,070 in which a tyrosine isreplaced by an alanine in the first position, U.S. Pat. No. 6,037,452,in which Factor XI is linked to an alkylene oxide group, and U.S. Pat.No. 6,046,380, in which the DNA encoding Factor IX is modified in atleast one splice site. As demonstrated herein, variants of Factor IX arewell known in the art, and the present disclosure encompasses thosevariants known or to be developed or discovered in the future.

Methods for determining the activity of a mutant or modified Factor IXcan be carried out using the methods described in the art, such as a onestage activated partial thromboplastin time assay as described in, forexample, Biggs (1972, Human Blood Coagulation Homeostasis and Thrombosis(Ed. 1), Oxford, Blackwell, Scientific, pg. 614). Briefly, to assay thebiological activity of a Factor IX molecule developed according to themethods of the present invention, the assay can be performed with equalvolumes of activated partial thromboplastin reagent, Factor IX deficientplasma isolated from a patient with hemophilia B using sterilephlebotomy techniques well known in the art, and normal pooled plasma asstandard, or the sample. In this assay, one unit of activity is definedas that amount present in one milliliter of normal pooled plasma.Further, an assay for biological activity based on the ability of FactorIX to reduce the clotting time of plasma from Factor IX-deficientpatients to normal can be performed as described in, for example,Proctor and Rapaport (Amer. J. Clin. Path. 36: 212 (1961).

The peptides of the invention include at least one N-linked or O-linkedglycosylation site, at least one of which is conjugated to a glycosylresidue that includes a PEG moiety. The PEG is covalently attached tothe peptide via an intact glycosyl linking group. The glycosyl linkinggroup is covalently attached to either an amino acid residue or aglycosyl residue of the peptide. Alternatively, the glycosyl linkinggroup is attached to one or more glycosyl units of a glycopeptide. Theinvention also provides conjugates in which the glycosyl linking groupis attached to both an amino acid residue and a glycosyl residue.

The PEG moiety is attached to an intact glycosyl linker directly, or viaa non-glycosyl linker, e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl.

Preferably, neither the amino nor the carboxy terminus of the Factor IXpeptide is derivatized with a polymeric modifying moiety.

The peptides of the invention include at least one N-linked or O-linkedglycosylation site, which is glycosylated with a glycosyl residue thatincludes a polymeric modifying moiety, e.g., a PEG moiety. In anexemplary embodiment, the PEG is covalently attached to the peptide viaan intact glycosyl linking group. The glycosyl linking group iscovalently attached to either an amino acid residue or a glycosylresidue of the peptide. Alternatively, the glycosyl linking group isattached to one or more glycosyl units of a glycopeptide. The inventionalso provides conjugates in which a glycosyl linking group is attachedto both an amino acid residue and a glycosyl residue.

The PEG moiety is attached to an intact glycosyl linker directly, or viaa non-glycosyl linker, e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl.

In an exemplary embodiment, the invention utilizes a modified sugaramine that has the formula:

in which J is a glycosyl moiety (e.g., a nucleotide sugar), L is a bondor a linker and R¹ is the modifying group, e.g., a polymeric modifyingmoiety. Exemplary bonds are those that are formed between an NH₂ moietyon the glycosyl moiety and a group of complementary reactivity on themodifying group. For example, when R¹ includes a carboxylic acid moiety,this moiety may be activated and coupled with the NH₂ moiety on theglycosyl residue affording a bond having the structure NHC(O)R¹. J ispreferably a glycosyl moiety that is “intact”, not having been degradedby exposure to conditions that cleave the pyranose or furanosestructure, e.g. oxidative conditions, e.g., sodium periodate.

Exemplary linkers include alkyl and heteroalkyl moieties. The linkersinclude linking groups, for example acyl-based linking groups, e.g.,—C(O)NH—, —OC(O)NH—, and the like. The linking groups are bonds formedbetween components of the species of the invention, e.g., between theglycosyl moiety and the linker (L), or between the linker and themodifying group (R¹). Other exemplary linking groups are ethers,thioethers and amines. For example, in one embodiment, the linker is anamino acid residue, such as a glycine residue. The carboxylic acidmoiety of the glycine is converted to the corresponding amide byreaction with an amine on the glycosyl residue, and the amine of theglycine is converted to the corresponding amide or urethane by reactionwith an activated carboxylic acid or carbonate of the modifying group.

Another exemplary linker is a PEG moiety, e.g., a PEG moiety that isfunctionalized with an amino acid residue. The PEG linker is conjugatedto the glycosyl group through the amino acid residue at one PEG terminusand bound to R¹ through the other PEG terminus. Alternatively, the aminoacid residue is bound to R¹ and the PEG terminus, which is not bound tothe amino acid, is bound to the glycosyl group.

An exemplary species of NH-L-R¹ has the formula:—NH{C(O)(CH₂)_(n)NH}_(s){C(O)(CH₂)_(b)(OCH₂CH₂)_(c)—O—(CH₂)_(d)NH}_(t)R¹,in which the indices s and t are independently 0 or 1. The indices a, band d are independently integers from 0 to 20, and c is an integer from1 to 2500. Other similar linkers are based on species in which an —NHmoiety is replaced by another group, for example, —S, —O or —CH₂. Asthose of skill will appreciate one or more of the bracketed moietiescorresponding to indices s and t can be replaced with a substituted orunsubstituted alkyl or heteroalkyl moiety.

More particularly, the invention utilizes compounds in which NH-L-R¹ is:NHC(O)(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)—O—(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)—O—(CH₂)_(d)NHR¹,NHC(O)O(CH₂)_(b)(OCH₂CH₂)_(c)—O—(CH₂)_(d)NHR¹,NH(CH₂)_(a)NHC(O)(CH₂)_(b)(OCH₂CH₂)_(c)—O—(CH₂)_(d)NHR¹,NHC(O)(CH₂)_(a)NHR¹, NH(CH₂)_(a)NHR¹, and NHR¹. In these formulae, theindices a, b and d are independently selected from the integers from 0to 20, preferably from 1 to 5. The index c is an integer from 1 to about2500.

In an exemplary embodiment, c is selected such that the PEG moiety isapproximately 1 kD, 5 kD, 10, kD, 15 kD, 20 kD or 30 kD.

In the discussion that follows, the invention is illustrated byreference to the use of selected derivatives of furanose and pyranose.Those of skill in the art will recognize that the focus of thediscussion is for clarity of illustration and that the structures andcompositions set forth are generally applicable across the genus ofsaccharide groups, modified saccharide groups, activated modifiedsaccharide groups and conjugates of modified saccharide groups.

In an exemplary embodiment, the invention provides a glycopeptide thatis conjugated to a polymeric modifying moiety through an intact glycosyllinking group having a formula that is selected from:

In Formulae I R² is H, CH₂OR⁷, COOR⁷ or OR⁷, in which R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl. When COOR⁷ is a carboxylic acid or carboxylate, both formsare represented by the designation of the single structure COO⁻ or COOH.In Formulae I and II, the symbols R³, R⁴, R⁵, R⁶ and R^(6′)independently represent H, substituted or unsubstituted alkyl, OR⁸,NHC(O)R⁹. The index d is 0 or 1. R⁸ and R⁹ are independently selectedfrom H, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, sialic acid or polysialic acid. At least one of R³, R⁴, R⁵,R⁶ or R^(6′) includes the polymeric modifying moiety e.g., PEG, linkedthrough a bond or a linking group. In an exemplary embodiment, R⁶ andR⁶, together with the carbon to which they are attached are componentsof the pyruvyl side chain of sialic acid. In a further exemplaryembodiment, this side chain is functionalized with the polymericmodifying moiety. In another exemplary embodiment, R⁶ and R^(6′),together with the carbon to which they are attached are components ofthe side chain of sialic acid and the polymeric modifying moiety is acomponent of R⁵.

In a further exemplary embodiment, the polymeric modifying moiety isbound to the sugar core, generally through a heteroatom, e.g, nitrogen,on the core through a linker, L, as shown below:

R¹ is the polymeric moiety and L is selected from a bond and a linkinggroup. The index w represents an integer selected from 1-6, preferably1-3 and more preferably 1-2. Exemplary linking groups includesubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl moieties and sialic acid. An exemplary component of thelinker is an acyl moiety.

An exemplary compound according to the invention has a structureaccording to Formulae I or II, in which at least one of R², R³, R⁴, R⁵,R⁶ or R^(6′) has the formula:

In another example according to this embodiment at least one of R², R³,R⁴, R⁵, R⁶ or R^(6′) has the formula:

in which s is an integer from 0 to 20 and R¹ is a linear polymericmodifying moiety.

In an exemplary embodiment, the polymeric modifying moiety-linkerconstruct is a branched structure that includes two or more polymericchains attached to central moiety. In this embodiment, the construct hasthe formula:

in which R¹ and L are as discussed above and w′ is an integer from 2 to6, preferably from 2 to 4 and more preferably from 2 to 3.

When L is a bond it is formed between a reactive functional group on aprecursor of R¹ and a reactive functional group of complementaryreactivity on the saccharyl core. When L is a non-zero order linker, aprecursor of L can be in place on the glycosyl moiety prior to reactionwith the R¹ precursor. Alternatively, the precursors of R¹ and L can beincorporated into a preformed cassette that is subsequently attached tothe glycosyl moiety. As set forth herein, the selection and preparationof precursors with appropriate reactive functional groups is within theability of those skilled in the art. Moreover, coupling the precursorsproceeds by chemistry that is well understood in the art.

In an exemplary embodiment, L is a linking group that is formed from anamino acid, or small peptide (e.g., 1-4 amino acid residues) providing amodified sugar in which the polymeric modifying moiety is attachedthrough a substituted alkyl linker. Exemplary linkers include glycine,lysine, serine and cysteine. The PEG moiety can be attached to the aminemoiety of the linker through an amide or urethane bond. The PEG islinked to the sulfur or oxygen atoms of cysteine and serine throughthioether or ether bonds, respectively.

In an exemplary embodiment, R⁵ includes the polymeric modifying moiety.In another exemplary embodiment, R⁵ includes both the polymericmodifying moiety and a linker, L, joining the modifying moiety to theremainder of the molecule. As discussed above, L can be a linear orbranched structure. Similarly, the polymeric modifying can be branchedor linear.

In one embodiment, the present invention provides an Factor IX peptidecomprising the moiety:

wherein D is a member selected from —OH and R¹-L-HN—; G is a memberselected from H and R¹-L- and —C(O)(C₁-C₆)alkyl; R¹ is a moietycomprising a straight-chain or branched poly(ethylene glycol) residue;and L is a linker, e.g., a bond (“zero order”), substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl. Inexemplary embodiments, when D is OH, G is R¹-L-, and when G is—C(O)(C₁-C₆)alkyl, D is R¹-L-NH—.

In another exemplary embodiment, the invention provides a conjugateformed between a modified sugar of the invention and a substrate FactorIX peptide. In this embodiment, the sugar moiety of the modified sugarbecomes a glycosyl linking group interposed between the peptidesubstrate and the modifying group. An exemplary glycosyl linking groupis an intact glycosyl linking group, in which the glycosyl moiety ormoieties forming the linking group are not degraded by chemical (e.g.,sodium metaperiodate) or enzymatic (e.g., oxidase) processes. Selectedconjugates of the invention include a modifying group that is attachedto the amine moiety of an amino-saccharide, e.g., mannosamine,glucosamine, galactosamine, sialic acid etc. Exemplary modifyinggroup-intact glycosyl linking group cassettes according to this motifare based on a sialic acid structure, such as those having the formulae:

In the formulae above, R¹ and L are as described above. Further detailabout the structure of exemplary R¹ groups is provided below.

In still a further exemplary embodiment, the conjugate is formed betweena substrate Factor IX and a saccharyl moiety in which the modifyinggroup is attached through a linker at the 6-carbon position of thesaccharyl moiety. Thus, illustrative conjugates according to thisembodiment have the formula:

in which the radicals are as discussed above. Such saccharyl moietiesinclude, without limitation, glucose, glucosamine, N-acetyl-glucosamine,galactose, galactosamine, N-acetyl-galactosamine, mannose, mannosamine,N-acetyl-mannosamine, and the like.

Due to the versatility of the methods available for modifying glycosylresidues on a therapeutic peptide such as Factor IX, the glycosylstructures on the peptide conjugates of the invention can havesubstantially any structure. Moreover, the glycans can be O-linked orN-linked. As exemplified in the discussion below, each of the pyranoseand furanose derivatives discussed above can be a component of aglycosyl moiety of a peptide.

The invention provides a modified Factor IX peptide that includes aglycosyl group having the formula:

In other embodiments, the group has the formula:

In a still further exemplary embodiment, the group has the formula:

In yet another embodiment, the group has the formula:

in which the index p represents and integer from 1 to 10; and c iseither 0 or 1.

In an exemplary embodiment according to each of the formulae set forthabove, the PEG-glycosyl linking group is attached at Serine 61 (Ser 61)of Factor IX.

In an exemplary embodiment, a glycoPEGylated Factor IX peptide of theinvention includes at least one N-linked glycosyl residue selected fromthe glycosyl residues set forth below:

In the formulae above, the index t is 0 or 1 and the index p is aninteger from 1 to 10. The symbol R¹⁵ represents H, OH (e.g., Gal-OH), asialyl moiety, a polymer modified sialyl moiety (i.e., glycosyl linkinggroup-polymeric modifying moiety (Sia-L-R¹)) or a sialyl moiety to whichis bound a polymer modified sialyl moiety (e.g., Sia-Sia-L-R¹)(“Sia-Sia^(p)”). Exemplary polymer modified saccharyl moieties have astructure according to Formulae I and II. An exemplary Factor IX peptideof the invention will include at least one glycan having a R^(15′) thatincludes a structure according to Formulae I or II. The oxygen, with theopen valence, of Formulae I and II is preferably attached through aglycosidic linkage to a carbon of a Gal or GalNAc moiety. In a furtherexemplary embodiment, the oxygen is attached to the carbon at position 3of a galactose residue. In an exemplary embodiment, the modified sialicacid is linked α2,3-to the galactose residue. In another exemplaryembodiment, the sialic acid is linked α2,6-to the galactose residue.

In another exemplary embodiment, the invention provides an Factor IXpeptide conjugate that includes a glycosyl linking group, such as thoseset forth above, that is covalently attached to an amino acid residue ofthe peptide. In one embodiment according to this motif, the glycosyllinking moiety is linked to a galactose residue through a Sia residue:

An exemplary species according to this motif is prepared by conjugatingSia-L-R¹ to a terminal sialic acid of a glycan using an enzyme thatforms Sia-Sia bonds, e.g., CST-II, ST8Sia-II, ST8Sia-III and ST8Sia-IV.

In another exemplary embodiment, the glycans have a formula that isselected from the group:

and combinations thereof.

The glycans of this group generally correspond to those found on anFactor IX peptide that is produced by insect (e.g., Sf-9) cells,following remodeling according to the methods set forth herein. Forexample insect-derived Factor IX that is expressed with a tri-mannosylcore is subsequently contacted with a GlcNAc donor and a GlcNActransferase and a Gal donor and a Gal transferase. Appending GlcNAc andGal to the tri-mannosyl core is accomplished in either two steps or asingle step. A modified sialic acid is added to at least one branch ofthe glycosyl moiety as discussed herein. Those Gal moieties that are notfunctionalized with the modified sialic acid are optionally “capped” byreaction with a sialic acid donor in the presence of a sialyltransferase.

In an exemplary embodiment, the glycosyl linking group is attached to amember selected from Asn 157, Asn 167 and combinations thereof.

In an exemplary embodiment, at least 60% of terminal Gal moieties in apopulation of peptides is capped with sialic acid, preferably at least70%, more preferably, at least 80%, still more preferably at least 90%and even more preferably at least 95%, 96%, 97%, 98% or 99% are cappedwith sialic acid.

In each of the formulae above, R¹⁵/R^(15′) is as discussed above.Moreover, an exemplary modified Factor IX peptide of the invention willinclude at least one glycan with an R¹⁵/R^(15′) moiety having astructure according to Formulae I or II.

In an exemplary embodiment, the glycosyl linking moiety has the formula:

in which b is 0 or 1. The index s represents and integer from 1 to 10;and f represents and integer from 1 to 2500. Generally preferred is theuse of a PEG moiety that has a molecular weight of about 20 kDa. Alsopreferred is the attachment of the glycosyl linking group a memberselected from Ser 61, Ser 63, Thr 159, Thr 169, Thr 172 and combinationsthereof.

In another exemplary embodiment, the Factor IX is derived from insectcells, remodeled by adding GlcNAc and Gal to the mannose core andglycopegylated using a sialic acid bearing a linear PEG moiety,affording an Factor IX peptide that comprises at least one moiety havingthe formula:

in which s represents and integer from 1 to 10; and f represents andinteger from 1 to 2500.

As discussed herein, the PEG of use in the conjugates of the inventioncan be linear or branched. An exemplary precursor of use to form thebranched conjugates according to this embodiment of the invention hasthe formula:

The branched polymer species according to this formula are essentiallypure water-soluble polymers. X^(3′) is a moiety that includes anionizable, e.g., OH, COOH, H₂PO₄, HSO₃, HPO₃, and salts thereof, etc.)or other reactive functional group, e.g., infra. C is carbon. X⁵ ispreferably a non-reactive group (e.g., H, unsubstituted alkyl,unsubstituted heteroalkyl), and can be a polymeric arm. R¹⁶ and R¹⁷ areindependently selected polymeric arms, e.g., nonpeptidic, nonreactivepolymeric arms (e.g., PEG)). X² and X⁴ are linkage fragments that arepreferably essentially non-reactive under physiological conditions,which may be the same or different. An exemplary linker includes neitheraromatic nor ester moieties. Alternatively, these linkages can includeone or more moiety that is designed to degrade under physiologicallyrelevant conditions, e.g., esters, disulfides, etc. X² and X⁴ joinpolymeric arms R¹⁶ and R¹⁷ to C. When X^(3′) is reacted with a reactivefunctional group of complementary reactivity on a linker, sugar orlinker-sugar cassette, X^(3′) is converted to a component of linkagefragment X³

Exemplary linkage fragments for X², X³ and X⁴ are independently selectedand include S, SC(O)NH, HNC(O)S, SC(O)O, O, NH, NHC(O), (O)CNH andNHC(O)O, and OC(O)NH, CH₂S, CH₂O, CH₂CH₂O, CH₂CH₂S, (CH₂)_(o)O,(CH₂)_(o)S or (CH₂)_(o)Y′-PEG wherein, Y′ is S, NH, NHC(O), C(O)NH,NHC(O)O, OC(O)NH, or O and o is an integer from 1 to 50. In an exemplaryembodiment, the linkage fragments X² and X⁴ are different linkagefragments.

In an exemplary embodiment, the precursor (III), or an activatedderivative thereof, is reacted with, and thereby bound to a sugar, anactivated sugar or a sugar nucleotide through a reaction between X^(3′)and a group of complementary reactivity on the sugar moiety, e.g., anamine. Alternatively, X^(3′) reacts with a reactive functional group ona precursor to linker, L. One or more of R², R³, R⁴, R⁵ R⁶ or R^(6′) ofFormulae I and II can include the branched polymeric modifying moiety,or this moiety bound through L.

In an exemplary embodiment, the moiety:

is the linker arm, L. In this embodiment, an exemplary linker is derivedfrom a natural or unnatural amino acid, amino acid analogue or aminoacid mimetic, or a small peptide formed from one or more such species.For example, certain branched polymers found in the compounds of theinvention have the formula:

X^(a) is a linkage fragment that is formed by the reaction of a reactivefunctional group, e.g., X^(3′), on a precursor of the branched polymericmodifying moiety and a reactive functional group on the sugar moiety, ora precursor to a linker. For example, when X^(3′) is a carboxylic acid,it can be activated and bound directly to an amine group pendent from anamino-saccharide (e.g., Sia, GalNH₂, GlcNH₂, ManNH₂, etc.), forming anX^(a) that is an amide. Additional exemplary reactive functional groupsand activated precursors are described hereinbelow. The index crepresents an integer from 1 to 10. The other symbols have the sameidentity as those discussed above.

In another exemplary embodiment, X^(a) is a linking moiety formed withanother linker:

in which X^(b) is a second linkage fragment and is independentlyselected from those groups set forth for X^(a), and, similar to L, L¹ isa bond, substituted or unsubstituted alkyl or substituted orunsubstituted heteroalkyl.

Exemplary species for X^(a) and X^(b) include S, SC(O)NH, HNC(O)S,SC(O)O, O, NH, NHC(O), C(O)NH and NHC(O)O, and OC(O)NH.

In another exemplary embodiment, X⁴ is a peptide bond to R¹⁷, which isan amino acid, di-peptide (e.g., Lys-Lys) or tri-peptide (E.G.,Lys-Lys-Lys) in which the alpha-amine moiety(ies) and/or side chainheteroatom(s) are modified with a polymeric modifying moiety.

In a further exemplary embodiment, the conjugates of the inventioninclude a moiety, e.g., an R¹⁵/R^(15′) moiety that has a formula that isselected from:

in which the identity of the radicals represented by the various symbolsis the same as that discussed hereinabove. L^(a) is a bond or a linkeras discussed above for L and L¹, e.g., substituted or unsubstitutedalkyl or substituted or unsubstituted heteroalkyl moiety. In anexemplary embodiment, L^(a) is a moiety of the side chain of sialic acidthat is functionalized with the polymeric modifying moiety as shown.Exemplary L^(a) moieties include substituted or unsubstituted alkylchains that include one or more OH or NH₂.

In yet another exemplary embodiment, the invention provides conjugateshaving a moiety, e.g., an R¹⁵/R^(15′) moiety with formula:

The identity of the radicals represented by the various symbols is thesame as that discussed hereinabove. As those of skill will appreciate,the linker arm in Formulae VI and VII is equally applicable to othermodified sugars set forth herein. In exemplary embodiment, the speciesof Formulae VI and VII are the R¹⁵ moieties attached to the glycanstructures set forth herein.

In yet another exemplary embodiment, the Factor IX peptide includes anR^(15′) moiety with the formula:

in which the identities of the radicals are as discussed above. Anexemplary species for L^(a) is —(CH₂)_(j)C(O)NH(CH₂)_(h)C(O)NH—, inwhich h and j are independently selected integers from 0 to 10. Afurther exemplary species is —C(O)NH—.

The embodiments of the invention set forth above are further exemplifiedby reference to species in which the polymer is a water-soluble polymer,particularly poly(ethylene glycol) (“PEG”), e.g., methoxy-poly(ethyleneglycol). Those of skill will appreciate that the focus in the sectionsthat follow is for clarity of illustration and the various motifs setforth using PEG as an exemplary polymer are equally applicable tospecies in which a polymer other than PEG is utilized.

PEG of any molecular weight, e.g. 1 kDa, 2 kDa, 5 kDa, 10 kDa, 15 kDa,20 kDa, 30 kDa and 40 kDa is of use in the present invention.

In an exemplary embodiment, the R¹⁵ moiety has a formula that is amember selected from the group:

In each of the structures above, the linker fragment —NH(CH₂)_(a)— canbe present or absent. In other exemplary embodiments, the conjugateincludes an R¹⁵ moiety selected from the group:

In each of the formulae above, the indices e and f are independentlyselected from the integers from 1 to 2500. In further exemplaryembodiments, e and f are selected to provide a PEG moiety that is about1 kD, 2 kD, 10 kD, 15 kD, 20 kD, 30 kD or 40 kD. The symbol Q representssubstituted or unsubstituted alkyl (e.g., C₁-C₆ alkyl, e.g., methyl),substituted or unsubstituted heteroalkyl or H.

Other branched polymers have structures based on di-lysine (Lys-Lys)peptides, e.g.:

and tri-lysine peptides (Lys-Lys-Lys), e.g.:

In each of the figures above, e, f, f′ and f″ represent integersindependently selected from 1 to 2500. The indices q, q′ and q″represent integers independently selected from 1 to 20.

In another exemplary embodiment, the Factor IX peptide comprises aglycosyl moiety selected from the formulae:

in which L^(a) is a bond or a linker as described herein; the index trepresents 0 or 1; and the index c represents 0 or 1. Each of thesegroups can be included as components of the mono-, bi-, tri- andtetra-antennary saccharide structures set forth above.

In yet another embodiment, the conjugates of the invention include amodified glycosyl residue that includes the substructure selected from:

in which the index a and the linker L^(a) are as discussed above. Theindex p is an integer from 1 to 10. The index c represents 0 or 1. Eachof these groups can be included as components of the mono-, bi-, tri-and tetra-antennary saccharide structures set forth above.

In a further exemplary embodiment, the invention utilizes modifiedsugars in which the 6-hydroxyl position is converted to thecorresponding amine moiety, which bears a linker-modifying groupcassette such as those set forth above. Exemplary saccharyl groups thatcan be used as the core of these modified sugars include Gal, GalNAc,Glc, GlcNAc, Fuc, Xyl, Man, and the like. A representative modifiedsugar according to this embodiment has the formula:

in which R¹¹-R¹⁴ are members independently selected from H, OH, C(O)CH₃,NH, and NH C(O)CH₃. R¹⁰ is a link to another glycosyl residue(—O-glycosyl) or to an amino acid of the Factor IX peptide (—NH-(FactorIX)). R¹⁴ is OR¹, NHR¹ or NH-L-R¹. R¹ and NH-L-R¹ are as describedabove.

Selected conjugates according to this motif are based on mannose,galactose or glucose, or on species having the stereochemistry ofmannose, galactose or glucose. The general formulae of these conjugatesare:

As discussed above, the invention provides saccharides bearing amodifying group, activated analogues of these species and conjugatesformed between species such as peptides and lipids and a modifiedsaccharide of the invention.

Modified Sugars

The present invention uses modified sugars and modified sugarnucleotides to form conjugates of the modified sugars. In modified sugarcompounds of use in the invention, the sugar moiety is preferably asaccharide, a deoxy-saccharide, an amino-saccharide, or an N-acylsaccharide. The term “saccharide” and its equivalents, “saccharyl,”“sugar,” and “glycosyl” refer to monomers, dimers, oligomers andpolymers. The sugar moiety is also functionalized with a modifyinggroup. The modifying group is conjugated to the sugar moiety, typically,through conjugation with an amine, sulfhydryl or hydroxyl, e.g., primaryhydroxyl, moiety on the sugar. In an exemplary embodiment, the modifyinggroup is attached through an amine moiety on the sugar, e.g., through anamide, a urethane or a urea that is formed through the reaction of theamine with a reactive derivative of the modifying group.

Any sugar can be utilized as the sugar core of the glycosyl linkinggroup of the conjugates of the invention. Exemplary sugar cores that areuseful in forming the compositions of the invention include, but are notlimited to, glucose, galactose, mannose, fucose, and sialic acid. Otheruseful sugars include amino sugars such as glucosamine, galactosamine,mannosamine, the 5-amine analogue of sialic acid and the like. The sugarcore can be a structure found in nature or it can be modified to providea site for conjugating the modifying group. For example, in oneembodiment, the invention provides a sialic acid derivative in which the9-hydroxy moiety is replaced with an amine. The amine is readilyderivatized with an activated analogue of a selected modifying group.

Exemplary modified sugars are modified with water-soluble orwater-insoluble polymers. Examples of useful polymer are furtherexemplified below.

Water-Soluble Polymers

Many water-soluble polymers are known to those of skill in the art andare useful in practicing the present invention. The term water-solublepolymer encompasses species such as saccharides (e.g., dextran, amylose,hyalouronic acid, poly(sialic acid), heparans, heparins, etc.); poly(amino acids), e.g., poly(aspartic acid) and poly(glutamic acid);nucleic acids; synthetic polymers (e.g., poly(acrylic acid),poly(ethers), e.g., poly(ethylene glycol); peptides, proteins, and thelike. The present invention may be practiced with any water-solublepolymer with the sole limitation that the polymer must include a pointat which the remainder of the conjugate can be attached.

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and more WO 93/15189, and for conjugation between activatedpolymers and peptides, e.g. Coagulation Factor VIII (WO 94/15625),hemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No.4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App.Biochem. Biotech. 11: 141-45 (1985)).

Preferred water-soluble polymers are those in which a substantialproportion of the polymer molecules in a sample of the polymer are ofapproximately the same molecular weight; such polymers are“homodisperse.”

The present invention is further illustrated by reference to apoly(ethylene glycol) conjugate. Several reviews and monographs on thefunctionalization and conjugation of PEG are available. See, forexample, Harris, Macronol. Chem. Phys. C25: 325-373 (1985); Scouten,Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb.Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky,Bioconjugate Chem. 6: 150-165 (1995); and Bhadra, et al., Pharmazie,57:5-29 (2002). Routes for preparing reactive PEG molecules and formingconjugates using the reactive molecules are known in the art. Forexample, U.S. Pat. No. 5,672,662 discloses a water soluble andisolatable conjugate of an active ester of a polymer acid selected fromlinear or branched poly(alkylene oxides), poly(oxyethylated polyols),poly(olefinic alcohols), and poly(acrylomorpholine).

U.S. Pat. No. 6,376,604 sets forth a method for preparing awater-soluble 1-benzotriazolylcarbonate ester of a water-soluble andnon-peptidic polymer by reacting a terminal hydroxyl of the polymer withdi(1-benzotriazoyl)carbonate in an organic solvent. The active ester isused to form conjugates with a biologically active agent such as aprotein or peptide.

WO 99/45964 describes a conjugate comprising a biologically active agentand an activated water soluble polymer comprising a polymer backbonehaving at least one terminus linked to the polymer backbone through astable linkage, wherein at least one terminus comprises a branchingmoiety having proximal reactive groups linked to the branching moiety,in which the biologically active agent is linked to at least one of theproximal reactive groups. Other branched poly(ethylene glycols) aredescribed in WO 96/21469, U.S. Pat. No. 5,932,462 describes a conjugateformed with a branched PEG molecule that includes a branched terminusthat includes reactive functional groups. The free reactive groups areavailable to react with a biologically active species, such as a proteinor peptide, forming conjugates between the poly(ethylene glycol) and thebiologically active species. U.S. Pat. No. 5,446,090 describes abifunctional PEG linker and its use in forming conjugates having apeptide at each of the PEG linker termini.

Conjugates that include degradable PEG linkages are described in WO99/34833; and WO 99/14259, as well as in U.S. Pat. No. 6,348,558. Suchdegradable linkages are applicable in the present invention.

The art-recognized methods of polymer activation set forth above are ofuse in the context of the present invention in the formation of thebranched polymers set forth herein and also for the conjugation of thesebranched polymers to other species, e.g., sugars, sugar nucleotides andthe like.

The modified sugars are prepared by reacting the glycosyl core (or alinker on the core) with a polymeric modifying moiety (or a linker onthe polymeric modifying moiety). The discussion that follows providesexamples of selected polymeric modifying moieties of use in theinvention. For example, representative polymeric modifying moietiesinclude structures that are based on side chain-containing amino acids,e.g., serine, cysteine, lysine, and small peptides, e.g., lys-lys.Exemplary structures include:

Those of skill will appreciate that the free amine in the di-lysinestructures can also be pegylated through an amide or urethane bond witha PEG moiety.

In yet another embodiment, the branched PEG moiety is based upon atri-lysine peptide. The tri-lysine can be mono-, di-, tri-, ortetra-PEG-ylated. Exemplary species according to this embodiment havethe formulae:

in which e, f and f′ are independently selected integers from 1 to 2500;and q, q′ and q″ are independently selected integers from 1 to 20.

As will be apparent to those of skill, the branched polymers of use inthe invention include variations on the themes set forth above. Forexample the di-lysine-PEG conjugate shown above can include threepolymeric subunits, the third bonded to the α-amine shown as unmodifiedin the structure above. Similarly, the use of a tri-lysinefunctionalized with three or four polymeric subunits labeled with thepolymeric modifying moiety in a desired manner is within the scope ofthe invention.

The polymeric modifying moieties can be activated for reaction with theglycosyl core. Exemplary structures of activated species (e.g.,carbonates and active esters) include:

Other activating, or leaving groups, appropriate for activating linearand branched PEGs of use in preparing the compounds set forth hereininclude, but are not limited to the species:

PEG molecules that are activated with these and other species andmethods of making the activated PEGs are set forth in WO 04/083259.

Those of skill in the art will appreciate that one or more of the m-PEGarms of the branched polymers shown above can be replaced by a PEGmoiety with a different terminus, e.g., OH, COOH, NH₂, C₂-C₁₀-alkyl,etc. Moreover, the structures above are readily modified by insertingalkyl linkers (or removing carbon atoms) between the α-carbon atom andthe functional group of the amino acid side chain. Thus, “homo”derivatives and higher homologues, as well as lower homologues arewithin the scope of cores for branched PEGs of use in the presentinvention.

The branched PEG species set forth herein are readily prepared bymethods such as that set forth in the scheme below:

in which X^(d) is O or S and r is an integer from 1 to 5. The indices eand f are independently selected integers from 1 to 2500. In anexemplary embodiment, one or both of these indices are selected suchthat the polymer is about 10 kD, 15 kD or 20 kD in molecular weight.

Thus, according to this scheme, a natural or unnatural amino acid iscontacted with an activated m-PEG derivative, in this case the tosylate,forming 1 by alkylating the side-chain heteroatom X^(d). Themono-functionalize m-PEG amino acid is submitted to N-acylationconditions with a reactive m-PEG derivative, thereby assembling branchedm-PEG 2. As one of skill will appreciate, the tosylate leaving group canbe replaced with any suitable leaving group, e.g., halogen, mesylate,triflate, etc. Similarly, the reactive carbonate utilized to acylate theamine can be replaced with an active ester, e.g., N-hydroxysuccinimide,etc., or the acid can be activated in situ using a dehydrating agentsuch as dicyclohexylcarbodiimide, carbonyldiimidazole, etc.

In other exemplary embodiments, the urea moiety is replaced by a groupsuch as a amide.

In an illustrative embodiment, the modified sugar is sialic acid andselected modified sugar compounds of use in the invention have theformulae:

The indices a, b and d are integers from 0 to 20. The index c is aninteger from 1 to 2500. The structures set forth above can be componentsof R¹⁵.

In another illustrative embodiment, a primary hydroxyl moiety of thesugar is functionalized with the modifying group. For example, the9-hydroxyl of sialic acid can be converted to the corresponding amineand functionalized to provide a compound according to the invention.Formulae according to this embodiment include:

The structures set forth above can be components of R¹⁵/R^(15′).

As those of skill in the art will appreciate, the sialic acid moiety inthe exemplary compounds above can be replaced with any otheramino-saccharide including, but not limited to, glucosamine,galactosamine, mannosamine, their N-acyl derivatives, and the like.

Although the present invention is exemplified in the preceding sectionsby reference to PEG, as those of skill will appreciate, an array ofpolymeric modifying moieties is of use in the compounds and methods setforth herein.

In selected embodiments, R¹ or L-R¹ is a branched PEG, for example, oneof the species set forth above. Illustrative modified sugars accordingto this embodiment include:

in which X⁴ is a bond or O. In each of the structures above, thealkylamine linker —(CH₂)aNH- can be present or absent. The structuresset forth above can be components of R¹⁵/R^(15′).

As discussed herein, the polymer-modified sialic acids of use in theinvention may also be linear structures. Thus, the invention providesfor conjugates that include a sialic acid moiety derived from astructure such as:

in which q and e are as discussed above.

Water-Insoluble Polymers

In another embodiment, analogous to those discussed above, the modifiedsugars include a water-insoluble polymer, rather than a water-solublepolymer. The conjugates of the invention may also include one or morewater-insoluble polymers. This embodiment of the invention isillustrated by the use of the conjugate as a vehicle with which todeliver a therapeutic peptide in a controlled manner. Polymeric drugdelivery systems are known in the art. See, for example, Dunn et al.,Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACS Symposium SeriesVol. 469, American Chemical Society, Washington, D.C. 1991. Those ofskill in the art will appreciate that substantially any known drugdelivery system is applicable to the conjugates of the presentinvention.

The motifs forth above for R¹, L-R¹, R¹⁵, R^(15′) and other radicals areequally applicable to water-insoluble polymers, which may beincorporated into the linear and branched structures without limitationutilizing chemistry readily accessible to those of skill in the art.

Representative water-insoluble polymers include, but are not limited to,polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates,polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, poly(methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate)polyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly (ethylene terephthalate), poly(vinyl acetate), polyvinylchloride, polystyrene, polyvinyl pyrrolidone, pluronics andpolyvinylphenol and copolymers thereof.

Synthetically modified natural polymers of use in conjugates of theinvention include, but are not limited to, alkyl celluloses,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, andnitrocelluloses. Particularly preferred members of the broad classes ofsynthetically modified natural polymers include, but are not limited to,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxymethyl cellulose, cellulose triacetate,cellulose sulfate sodium salt, and polymers of acrylic and methacrylicesters and alginic acid.

These and the other polymers discussed herein can be readily obtainedfrom commercial sources such as Sigma Chemical Co. (St. Louis, Mo.),Polysciences (Warrenton, Pa.), Aldrich (Milwaukee, Wis.), Fluka(Ronkonkoma, N.Y.), and BioRad (Richmond, Calif.), or else synthesizedfrom monomers obtained from these suppliers using standard techniques.

Representative biodegradable polymers of use in the conjugates of theinvention include, but are not limited to, polylactides, polyglycolidesand copolymers thereof, poly(ethylene terephthalate), poly(butyricacid), poly(valeric acid), poly(lactide-co-caprolactone),poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends andcopolymers thereof. Of particular use are compositions that form gels,such as those including collagen, pluronics and the like.

The polymers of use in the invention include “hybrid” polymers thatinclude water-insoluble materials having within at least a portion oftheir structure, a bioresorbable molecule. An example of such a polymeris one that includes a water-insoluble copolymer, which has abioresorbable region, a hydrophilic region and a plurality ofcrosslinkable functional groups per polymer chain.

For purposes of the present invention, “water-insoluble materials”includes materials that are substantially insoluble in water orwater-containing environments. Thus, although certain regions orsegments of the copolymer may be hydrophilic or even water-soluble, thepolymer molecule, as a whole, does not to any substantial measuredissolve in water.

For purposes of the present invention, the term “bioresorbable molecule”includes a region that is capable of being metabolized or broken downand resorbed and/or eliminated through normal excretory routes by thebody. Such metabolites or break down products are preferablysubstantially non-toxic to the body.

The bioresorbable region may be either hydrophobic or hydrophilic, solong as the copolymer composition as a whole is not renderedwater-soluble. Thus, the bioresorbable region is selected based on thepreference that the polymer, as a whole, remains water-insoluble.Accordingly, the relative properties, i.e., the kinds of functionalgroups contained by, and the relative proportions of the bioresorbableregion, and the hydrophilic region are selected to ensure that usefulbioresorbable compositions remain water-insoluble.

Exemplary resorbable polymers include, for example, syntheticallyproduced resorbable block copolymers of poly(α-hydroxy-carboxylicacid)/poly(oxyalkylene, (see, Cohn et al., U.S. Pat. No. 4,826,945).These copolymers are not crosslinked and are water-soluble so that thebody can excrete the degraded block copolymer compositions. See, Youneset al., J. Biomed. Mater. Res. 21: 1301-1316 (1987); and Cohn et al., J.Biomed. Mater. Res. 22: 993-1009 (1988).

Presently preferred bioresorbable polymers include one or morecomponents selected from poly(esters), poly(hydroxy acids),poly(lactones), poly(amides), poly(ester-amides), poly (amino acids),poly(anhydrides), poly(orthoesters), poly(carbonates),poly(phosphazines), poly(phosphoesters), poly(thioesters),polysaccharides and mixtures thereof. More preferably still, thebiosresorbable polymer includes a poly(hydroxy) acid component. Of thepoly(hydroxy) acids, polylactic acid, polyglycolic acid, polycaproicacid, polybutyric acid, polyvaleric acid and copolymers and mixturesthereof are preferred.

In addition to forming fragments that are absorbed in vivo(“bioresorbed”), preferred polymeric coatings for use in the methods ofthe invention can also form an excretable and/or metabolizable fragment.

Higher order copolymers can also be used in the present invention. Forexample, Casey et al., U.S. Pat. No. 4,438,253, which issued on Mar. 20,1984, discloses tri-block copolymers produced from thetransesterification of poly(glycolic acid) and an hydroxyl-endedpoly(alkylene glycol). Such compositions are disclosed for use asresorbable monofilament sutures. The flexibility of such compositions iscontrolled by the incorporation of an aromatic orthocarbonate, such astetra-p-tolyl orthocarbonate into the copolymer structure.

Other polymers based on lactic and/or glycolic acids can also beutilized. For example, Spinu, U.S. Pat. No. 5,202,413, which issued onApr. 13, 1993, discloses biodegradable multi-block copolymers havingsequentially ordered blocks of polylactide and/or polyglycolide producedby ring-opening polymerization of lactide and/or glycolide onto eitheran oligomeric diol or a diamine residue followed by chain extension witha di-functional compound, such as, a diisocyanate, diacylchloride ordichlorosilane.

Bioresorbable regions of coatings useful in the present invention can bedesigned to be hydrolytically and/or enzymatically cleavable. Forpurposes of the present invention, “hydrolytically cleavable” refers tothe susceptibility of the copolymer, especially the bioresorbableregion, to hydrolysis in water or a water-containing environment.Similarly, “enzymatically cleavable” as used herein refers to thesusceptibility of the copolymer, especially the bioresorbable region, tocleavage by endogenous or exogenous enzymes.

When placed within the body, the hydrophilic region can be processedinto excretable and/or metabolizable fragments. Thus, the hydrophilicregion can include, for example, polyethers, polyalkylene oxides,polyols, poly(vinyl pyrrolidine), poly(vinyl alcohol), poly(alkyloxazolines), polysaccharides, carbohydrates, peptides, proteins andcopolymers and mixtures thereof. Furthermore, the hydrophilic region canalso be, for example, a poly(alkylene) oxide. Such poly(alkylene) oxidescan include, for example, poly(ethylene) oxide, poly(propylene) oxideand mixtures and copolymers thereof.

Polymers that are components of hydrogels are also useful in the presentinvention. Hydrogels are polymeric materials that are capable ofabsorbing relatively large quantities of water. Examples of hydrogelforming compounds include, but are not limited to, polyacrylic acids,sodium carboxymethylcellulose, polyvinyl alcohol, polyvinyl pyrrolidine,gelatin, carrageenan and other polysaccharides,hydroxyethylenemethacrylic acid (HEMA), as well as derivatives thereof,and the like. Hydrogels can be produced that are stable, biodegradableand bioresorbable. Moreover, hydrogel compositions can include subunitsthat exhibit one or more of these properties.

Bio-compatible hydrogel compositions whose integrity can be controlledthrough crosslinking are known and are presently preferred for use inthe methods of the invention. For example, Hubbell et al., U.S. Pat.Nos. 5,410,016, which issued on Apr. 25, 1995 and 5,529,914, whichissued on Jun. 25, 1996, disclose water-soluble systems, which arecrosslinked block copolymers having a water-soluble central blocksegment sandwiched between two hydrolytically labile extensions. Suchcopolymers are further end-capped with photopolymerizable acrylatefunctionalities. When crosslinked, these systems become hydrogels. Thewater soluble central block of such copolymers can include poly(ethyleneglycol); whereas, the hydrolytically labile extensions can be apoly(α-hydroxy acid), such as polyglycolic acid or polylactic acid. See,Sawhney et al., Macromolecules 26: 581-587 (1993).

In another preferred embodiment, the gel is a thermoreversible gel.Thermoreversible gels including components, such as pluronics, collagen,gelatin, hyalouronic acid, polysaccharides, polyurethane hydrogel,polyurethane-urea hydrogel and combinations thereof are presentlypreferred.

In yet another exemplary embodiment, the conjugate of the inventionincludes a component of a liposome. Liposomes can be prepared accordingto methods known to those skilled in the art, for example, as describedin Eppstein et al., U.S. Pat. No. 4,522,811. For example, liposomeformulations may be prepared by dissolving appropriate lipid(s) (such asstearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline,arachadoyl phosphatidyl choline, and cholesterol) in an inorganicsolvent that is then evaporated, leaving behind a thin film of driedlipid on the surface of the container. An aqueous solution of the activecompound or its pharmaceutically acceptable salt is then introduced intothe container. The container is then swirled by hand to free lipidmaterial from the sides of the container and to disperse lipidaggregates, thereby forming the liposomal suspension.

The above-recited microparticles and methods of preparing themicroparticles are offered by way of example and they are not intendedto define the scope of microparticles of use in the present invention.It will be apparent to those of skill in the art that an array ofmicroparticles, fabricated by different methods, is of use in thepresent invention.

The structural formats discussed above in the context of thewater-soluble polymers, both straight-chain and branched are generallyapplicable with respect to the water-insoluble polymers as well. Thus,for example, the cysteine, serine, dilysine, and trilysine branchingcores can be functionalized with two water-insoluble polymer moieties.The methods used to produce these species are generally closelyanalogous to those used to produce the water-soluble polymers.

The Methods

In addition to the conjugates discussed above, the present inventionprovides methods for preparing these and other conjugates. Moreover, theinvention provides methods of preventing, curing or ameliorating adisease state by administering a conjugate of the invention to a subjectat risk of developing the disease or a subject that has the disease.

In exemplary embodiments, the conjugate is formed between a polymericmodifying moiety and a glycosylated or non-glycosylated peptide. Thepolymer is conjugated to the peptide via a glycosyl linking group, whichis interposed between, and covalently linked to both the peptide (orglycosyl residue) and the modifying group (e.g., water-soluble polymer).The method includes contacting the peptide with a mixture containing amodified sugar and an enzyme, e.g., a glycosyltransferase thatconjugates the modified sugar to the substrate. The reaction isconducted under conditions appropriate to form a covalent bond betweenthe modified sugar and the peptide. The sugar moiety of the modifiedsugar is preferably selected from nucleotide sugars.

In an exemplary embodiment, the modified sugar, such as those set forthabove, is activated as the corresponding nucleotide sugars. Exemplarysugar nucleotides that are used in the present invention in theirmodified form include nucleotide mono-, di- or triphosphates or analogsthereof. In a preferred embodiment, the modified sugar nucleotide isselected from a UDP-glycoside, CMP-glycoside, or a GDP-glycoside. Evenmore preferably, the sugar nucleotide portion of the modified sugarnucleotide is selected from UDP-galactose, UDP-galactosamine,UDP-glucose, UDP-glucosamine, GDP-mannose, GDP-fucose, CMP-sialic acid,or CMP-NeuAc. In an exemplary embodiment, the nucleotide phosphate isattached to C-1.

Thus, in an illustrative embodiment in which the glycosyl moiety issialic acid, the method of the invention utilizes compounds having theformulae:

in which L-R¹ is as discussed above, and L¹-R¹ represents a linker boundto the modifying group. As with L, exemplary linker species according toL¹ include a bond, alkyl or heteroalkyl moieties.

Moreover, as discussed above, the present invention provides for the useof nucleotide sugars that are modified with a water-soluble polymer,which is either straight-chain or branched. For example, compoundshaving the formula shown below are of use to prepare conjugates withinthe scope of the present invention:

in which X⁴ is O or a bond.

The invention also provides for the use of sugar nucleotides modifiedwith L-R¹ at the 6-carbon position. Exemplary species according to thisembodiment include:

in which the R groups, and L, represent moieties as discussed above. Theindex “y” is 0, 1 or 2. In an exemplary embodiment, L is a bond betweenNH and R¹. The base is a nucleic acid base.

Exemplary nucleotide sugars of use in the invention in which the carbonat the 6-position is modified include species having the stereochemistryof GDP mannose, e.g.:

in which X⁵ is a bond or O. The index i represents 0 or 1. The index arepresents an integer from 1 to 20. The indices e and f independentlyrepresent integers from 1 to 2500. Q, as discussed above, is H orsubstituted or unsubstituted C₁-C₆ alkyl. As those of skill willappreciate, the serine derivative, in which S is replaced with 0 alsofalls within this general motif.

In a still further exemplary embodiment, the invention provides aconjugate in which the modified sugar is based on the stereochemistry ofUDP galactose. An exemplary nucleotide sugar of use in this inventionhas the structure:

In another exemplary embodiment, the nucleotide sugar is based on thestereochemistry of glucose. Exemplary species according to thisembodiment have the formulae:

In general, the sugar moiety or sugar moiety-linker cassette and the PEGor PEG-linker cassette groups are linked together through the use ofreactive groups, which are typically transformed by the linking processinto a new organic functional group or unreactive species. The sugarreactive functional group(s), is located at any position on the sugarmoiety. Reactive groups and classes of reactions useful in practicingthe present invention are generally those that are well known in the artof bioconjugate chemistry. Currently favored classes of reactionsavailable with reactive sugar moieties are those, which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Useful reactive functional groups pendent from a sugar nucleus ormodifying group include, but are not limited to:

-   -   (a) carboxyl groups and various derivatives thereof including,        but not limited to, N-hydroxysuccinimide esters,        N-hydroxybenztriazole esters, acid halides, acyl imidazoles,        thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl and        aromatic esters;    -   (b) hydroxyl groups, which can be converted to, e.g., esters,        ethers, aldehydes, etc.    -   (c) haloalkyl groups, wherein the halide can be later displaced        with a nucleophilic group such as, for example, an amine, a        carboxylate anion, thiol anion, carbanion, or an alkoxide ion,        thereby resulting in the covalent attachment of a new group at        the functional group of the halogen atom;    -   (d) dienophile groups, which are capable of participating in        Diels-Alder reactions such as, for example, maleimido groups;    -   (e) aldehyde or ketone groups, such that subsequent        derivatization is possible via formation of carbonyl derivatives        such as, for example, imines, hydrazones, semicarbazones or        oximes, or via such mechanisms as Grignard addition or        alkyllithium addition;    -   (f) sulfonyl halide groups for subsequent reaction with amines,        for example, to form sulfonamides;    -   (g) thiol groups, which can be, for example, converted to        disulfides or reacted with acyl halides;    -   (h) amine or sulfhydryl groups, which can be, for example,        acylated, alkylated or oxidized;    -   (i) alkenes, which can undergo, for example, cycloadditions,        acylation, Michael addition, etc; and    -   (j) epoxides, which can react with, for example, amines and        hydroxyl compounds.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive sugar nucleus or modifying group. Alternatively, a reactivefunctional group can be protected from participating in the reaction bythe presence of a protecting group. Those of skill in the art understandhow to protect a particular functional group such that it does notinterfere with a chosen set of reaction conditions. For examples ofuseful protecting groups, see, for example, Greene et al., PROTECTIVEGROUPS IN ORGANIC SYNTHESIS, John Wiley & Sons, New York, 1991.

In the discussion that follows, a number of specific examples ofmodified sugars that are useful in practicing the present invention areset forth. In the exemplary embodiments, a sialic acid derivative isutilized as the sugar nucleus to which the modifying group is attached.The focus of the discussion on sialic acid derivatives is for clarity ofillustration only and should not be construed to limit the scope of theinvention. Those of skill in the art will appreciate that a variety ofother sugar moieties can be activated and derivatized in a manneranalogous to that set forth using sialic acid as an example. Forexample, numerous methods are available for modifying galactose,glucose, N-acetylgalactosamine and fucose to name a few sugarsubstrates, which are readily modified by art recognized methods. See,for example, Elhalabi et al., Curr. Med. Chem. 6: 93 (1999); and Schaferet al., J. Org. Chem. 65: 24 (2000)).

In an exemplary embodiment, the modified sugar is based upon a6-amino-N-acetyl-glycosyl moiety. As shown in FIG. 5 forN-acetylgalactosamine, the 6-amino-sugar moiety is readily prepared bystandard methods.

In the scheme above, the index n represents an integer from 1 to 2500.In an exemplary embodiment, this index is selected such that the polymeris about 10 kD, 15 kD or 20 kD in molecular weight. The symbol “A”represents an activating group, e.g., a halo, a component of anactivated ester (e.g., a N-hydroxysuccinimide ester), a component of acarbonate (e.g., p-nitrophenyl carbonate) and the like. Those of skillin the art will appreciate that other PEG-amide nucleotide sugars arereadily prepared by this and analogous methods.

The acceptor peptide is typically synthesized de novo, or recombinantlyexpressed in a prokaryotic cell (e.g., bacterial cell, such as E. coli)or in a eukaryotic cell such as a mammalian, yeast, insect, fungal orplant cell. The peptide can be either a full-length protein or afragment. Moreover, the peptide can be a wild type or mutated peptide.In an exemplary embodiment, the peptide includes a mutation that addsone or more N- or O-linked glycosylation sites to the peptide sequence.

The method of the invention also provides for modification ofincompletely glycosylated peptides that are produced recombinantly. Manyrecombinantly produced glycoproteins are incompletely glycosylated,exposing carbohydrate residues that may have undesirable properties,e.g., immunogenicity, recognition by the RES. Employing a modified sugarin a method of the invention, the peptide can be simultaneously furtherglycosylated and derivatized with, e.g., a water-soluble polymer,therapeutic agent, or the like. The sugar moiety of the modified sugarcan be the residue that would properly be conjugated to the acceptor ina fully glycosylated peptide, or another sugar moiety with desirableproperties.

Those of skill will appreciate that the invention can be practiced usingsubstantially any peptide or glycopeptide from any source. Exemplarypeptides with which the invention can be practiced are set forth in WO03/031464, and the references set forth therein.

Peptides modified by the methods of the invention can be synthetic orwild-type peptides or they can be mutated peptides, produced by methodsknown in the art, such as site-directed mutagenesis. Glycosylation ofpeptides is typically either N-linked or O-linked. An exemplaryN-linkage is the attachment of the modified sugar to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of a carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-linked glycosylation refers to the attachment ofone sugar (e.g., N-acetylgalactosamine, galactose, mannose, GlcNAc,glucose, fucose or xylose) to the hydroxy side chain of a hydroxyaminoacid, preferably serine or threonine, although unusual or non-naturalamino acids, e.g., 5-hydroxyproline or 5-hydroxylysine may also be used.

Moreover, in addition to peptides, the methods of the present inventioncan be practiced with other biological structures (e.g., glycolipids,lipids, sphingoids, ceramides, whole cells, and the like, containing aglycosylation site).

Addition of glycosylation sites to a peptide or other structure isconveniently accomplished by altering the amino acid sequence such thatit contains one or more glycosylation sites. The addition may also bemade by the incorporation of one or more species presenting an —OHgroup, preferably serine or threonine residues, within the sequence ofthe peptide (for O-linked glycosylation sites). The addition may be madeby mutation or by full chemical synthesis of the peptide. The peptideamino acid sequence is preferably altered through changes at the DNAlevel, particularly by mutating the DNA encoding the peptide atpreselected bases such that codons are generated that will translateinto the desired amino acids. The DNA mutation(s) are preferably madeusing methods known in the art.

In an exemplary embodiment, the glycosylation site is added by shufflingpolynucleotides. Polynucleotides encoding a candidate peptide can bemodulated with DNA shuffling protocols. DNA shuffling is a process ofrecursive recombination and mutation, performed by random fragmentationof a pool of related genes, followed by reassembly of the fragments by apolymerase chain reaction-like process. See, e.g., Stemmer, Proc. Natl.Acad. Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391(1994); and U.S. Pat. Nos. 5,605,793, 5,837,458, 5,830,721 and5,811,238.

Exemplary peptides with which the present invention can be practiced,methods of adding or removing glycosylation sites, and adding orremoving glycosyl structures or substructures are described in detail inWO03/031464 and related U.S. and PCT applications.

The present invention also takes advantage of adding to (or removingfrom) a peptide one or more selected glycosyl residues, after which amodified sugar is conjugated to at least one of the selected glycosylresidues of the peptide. The present embodiment is useful, for example,when it is desired to conjugate the modified sugar to a selectedglycosyl residue that is either not present on a peptide or is notpresent in a desired amount. Thus, prior to coupling a modified sugar toa peptide, the selected glycosyl residue is conjugated to the peptide byenzymatic or chemical coupling. In another embodiment, the glycosylationpattern of a glycopeptide is altered prior to the conjugation of themodified sugar by the removal of a carbohydrate residue from theglycopeptide. See, for example WO 98/31826.

Addition or removal of any carbohydrate moieties present on theglycopeptide is accomplished either chemically or enzymatically. Anexemplary chemical deglycosylation is brought about by exposure of thepolypeptide variant to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the peptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259: 52 (1987) and by Edge et al., Anal Biochem. 118: 131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptidevariants can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol. 138:350 (1987).

In an exemplary embodiment, the peptide is essentially completelydesialylated with neuraminidase prior to performing glycoconjugation orremodeling steps on the peptide. Following the glycoconjugation orremodeling, the peptide is optionally re-sialylated using asialyltransferase. In an exemplary embodiment, the re-sialylation occursat essentially each (e.g., >80%, preferably greater than 85%, greaterthan 90%, preferably greater than 95% and more preferably greater than96%, 97%, 98% or 99%) terminal saccharyl acceptor in a population ofsialyl acceptors. In a preferred embodiment, the saccharide has asubstantially uniform sialylation pattern (i.e., substantially uniformglycosylation pattern).

Chemical addition of glycosyl moieties is carried out by anyart-recognized method. Enzymatic addition of sugar moieties ispreferably achieved using a modification of the methods set forthherein, substituting native glycosyl units for the modified sugars usedin the invention. Other methods of adding sugar moieties are disclosedin U.S. Pat. Nos. 5,876,980, 6,030,815, 5,728,554, and 5,922,577.

Exemplary attachment points for selected glycosyl residue include, butare not limited to: (a) consensus sites for N-linked glycosylation, andsites for O-linked glycosylation; (b) terminal glycosyl moieties thatare acceptors for a glycosyltransferase; (c) arginine, asparagine andhistidine; (d) free carboxyl groups; (e) free sulfhydryl groups such asthose of cysteine; (f) free hydroxyl groups such as those of serine,threonine, or hydroxyproline; (g) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan; or (h) the amide group ofglutamine. Exemplary methods of use in the present invention aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC CRIT. REV. BIOCHEM., pp. 259-306 (1981).

In one embodiment, the invention provides a method for linking two ormore peptides through a linking group. The linking group is of anyuseful structure and may be selected from straight- and branched-chainstructures. Preferably, each terminus of the linker, which is attachedto a peptide, includes a modified sugar (i.e., a nascent intact glycosyllinking group).

In an exemplary method of the invention, two peptides are linkedtogether via a linker moiety that includes a polymeric (e.g., PEGlinker). The construct conforms to the general structure set forth inthe cartoon above. As described herein, the construct of the inventionincludes two intact glycosyl linking groups (i.e., s+t=1). The focus ona PEG linker that includes two glycosyl groups is for purposes ofclarity and should not be interpreted as limiting the identity of linkerarms of use in this embodiment of the invention.

Thus, a PEG moiety is functionalized at a first terminus with a firstglycosyl unit and at a second terminus with a second glycosyl unit. Thefirst and second glycosyl units are preferably substrates for differenttransferases, allowing orthogonal attachment of the first and secondpeptides to the first and second glycosyl units, respectively. Inpractice, the (glycosyl)¹-PEG-(glycosyl)² linker is contacted with thefirst peptide and a first transferase for which the first glycosyl unitis a substrate, thereby forming (peptide)¹-(glycosyl)¹-PEG-(glycosyl)².Transferase and/or unreacted peptide is then optionally removed from thereaction mixture. The second peptide and a second transferase for whichthe second glycosyl unit is a substrate are added to the(peptide)¹-(glycosyl)¹-PEG-(glycosyl)² conjugate, forming(peptide)¹-(glycosyl)¹-PEG-(glycosyl)²-(peptide)²; at least one of theglycosyl residues is either directly or indirectly O-linked. Those ofskill in the art will appreciate that the method outlined above is alsoapplicable to forming conjugates between more than two peptides by, forexample, the use of a branched PEG, dendrimer, poly(amino acid),polysaccharide or the like.

In an exemplary embodiment, the peptide that is modified by a method ofthe invention is a glycopeptide that is produced in mammalian cells(e.g., CHO cells) or in a transgenic animal and thus, contains N- and/orO-linked oligosaccharide chains, which are incompletely sialylated. Theoligosaccharide chains of the glycopeptide lacking a sialic acid andcontaining a terminal galactose residue can be PEGylated, PPGylated orotherwise modified with a modified sialic acid.

In Scheme 1, the amino glycoside 1, is treated with the active ester ofa protected amino acid (e.g., glycine) derivative, converting the sugaramine residue into the corresponding protected amino acid amide adduct.The adduct is treated with an aldolase to form α-hydroxy carboxylate 2.Compound 2 is converted to the corresponding CMP derivative by theaction of CMP-SA synthetase, followed by catalytic hydrogenation of theCMP derivative to produce compound 3. The amine introduced via formationof the glycine adduct is utilized as a locus of PEG attachment byreacting compound 3 with an activated PEG or PPG derivative (e.g.,PEG-C(O)NHS, PEG-OC(O)O-p-nitrophenyl), producing species such as 4 or5, respectively.

Conjugation of Modified Sugars to Peptides

The PEG modified sugars are conjugated to a glycosylated ornon-glycosylated peptide using an appropriate enzyme to mediate theconjugation. Preferably, the concentrations of the modified donorsugar(s), enzyme(s) and acceptor peptide(s) are selected such thatglycosylation proceeds until the acceptor is consumed. Theconsiderations discussed below, while set forth in the context of asialyltransferase, are generally applicable to other glycosyltransferasereactions.

A number of methods of using glycosyltransferases to synthesize desiredoligosaccharide structures are known and are generally applicable to theinstant invention. Exemplary methods are described, for instance, WO96/32491, Ito et al., Pure Appl. Chem. 65: 753 (1993), U.S. Pat. Nos.5,352,670, 5,374,541, 5,545,553, commonly owned U.S. Pat. Nos.6,399,336, and 6,440,703, and commonly owned published PCT applications,WO 03/031464, WO 04/033651, WO 04/099231, which are incorporated hereinby reference.

The present invention is practiced using a single glycosyltransferase ora combination of glycosyltransferases. For example, one can use acombination of a sialyltransferase and a galactosyltransferase. In thoseembodiments using more than one enzyme, the enzymes and substrates arepreferably combined in an initial reaction mixture, or the enzymes andreagents for a second enzymatic reaction are added to the reactionmedium once the first enzymatic reaction is complete or nearly complete.By conducting two enzymatic reactions in sequence in a single vessel,overall yields are improved over procedures in which an intermediatespecies is isolated. Moreover, cleanup and disposal of extra solventsand by-products is reduced.

In a preferred embodiment, each of the first and second enzyme is aglycosyltransferase. In another preferred embodiment, one enzyme is anendoglycosidase. In an additional preferred embodiment, more than twoenzymes are used to assemble the modified glycoprotein of the invention.The enzymes are used to alter a saccharide structure on the peptide atany point either before or after the addition of the modified sugar tothe peptide.

In another embodiment, the method makes use of one or more exo- orendoglycosidase. The glycosidase is typically a mutant, which isengineered to form glycosyl bonds rather than rupture them. The mutantglycanase typically includes a substitution of an amino acid residue foran active site acidic amino acid residue. For example, when theendoglycanase is endo-H, the substituted active site residues willtypically be Asp at position 130, Glu at position 132 or a combinationthereof. The amino acids are generally replaced with serine, alanine,asparagine, or glutamine.

The mutant enzyme catalyzes the reaction, usually by a synthesis stepthat is analogous to the reverse reaction of the endoglycanasehydrolysis step. In these embodiments, the glycosyl donor molecule(e.g., a desired oligo- or mono-saccharide structure) contains a leavinggroup and the reaction proceeds with the addition of the donor moleculeto a GlcNAc residue on the protein. For example, the leaving group canbe a halogen, such as fluoride. In other embodiments, the leaving groupis a Asn, or a Asn-peptide moiety. In further embodiments, the GlcNAcresidue on the glycosyl donor molecule is modified. For example, theGlcNAc residue may comprise a 1,2 oxazoline moiety.

In a preferred embodiment, each of the enzymes utilized to produce aconjugate of the invention are present in a catalytic amount. Thecatalytic amount of a particular enzyme varies according to theconcentration of that enzyme's substrate as well as to reactionconditions such as temperature, time and pH value. Means for determiningthe catalytic amount for a given enzyme under preselected substrateconcentrations and reaction conditions are well known to those of skillin the art.

The temperature at which an above process is carried out can range fromjust above freezing to the temperature at which the most sensitiveenzyme denatures. Preferred temperature ranges are about 0° C. to about55° C., and more preferably about 20° C. to about 37° C. In anotherexemplary embodiment, one or more components of the present method areconducted at an elevated temperature using a thermophilic enzyme.

The reaction mixture is maintained for a period of time sufficient forthe acceptor to be glycosylated, thereby forming the desired conjugate.Some of the conjugate can often be detected after a few h, withrecoverable amounts usually being obtained within 24 h or less. Those ofskill in the art understand that the rate of reaction is dependent on anumber of variable factors (e.g, enzyme concentration, donorconcentration, acceptor concentration, temperature, solvent volume),which are optimized for a selected system.

The present invention also provides for the industrial-scale productionof modified peptides. As used herein, an industrial scale generallyproduces at least one gram of finished, purified conjugate.

In the discussion that follows, the invention is exemplified by theconjugation of modified sialic acid moieties to a glycosylated peptide.The exemplary modified sialic acid is labeled with PEG. The focus of thefollowing discussion on the use of PEG-modified sialic acid andglycosylated peptides is for clarity of illustration and is not intendedto imply that the invention is limited to the conjugation of these twopartners. One of skill understands that the discussion is generallyapplicable to the additions of modified glycosyl moieties other thansialic acid. Moreover, the discussion is equally applicable to themodification of a glycosyl unit with agents other than PEG includingother PEG moieties, therapeutic moieties, and biomolecules.

An enzymatic approach can be used for the selective introduction ofPEGylated or PPGylated carbohydrates onto a peptide or glycopeptide. Themethod utilizes modified sugars containing PEG, PPG, or a maskedreactive functional group, and is combined with the appropriateglycosyltransferase or glycosynthase. By selecting theglycosyltransferase that will make the desired carbohydrate linkage andutilizing the modified sugar as the donor substrate, the PEG or PPG canbe introduced directly onto the peptide backbone, onto existing sugarresidues of a glycopeptide or onto sugar residues that have been addedto a peptide.

In an exemplary embodiment, an acceptor for a sialyltransferase ispresent on the peptide to be modified either as a naturally occurringstructure or it is placed there recombinantly, enzymatically orchemically. Suitable acceptors, include, for example, galactosylacceptors such as Galβ1,4GlcNAc, Galβ1,4GalNAc, Galβ1,3GalNAc,lacto-N-tetraose, Galβ1,3GlcNAc, Galβ1,3Ara, Galβ1,6GlcNAc, Galβ1,4Glc(lactose), and other acceptors known to those of skill in the art (see,e.g., Paulson et al., J. Biol. Chem. 253: 5617-5624 (1978)). Exemplarysialyltransferases are set forth herein.

In one embodiment, an acceptor for the sialyltransferase is present onthe glycopeptide to be modified upon in vivo synthesis of theglycopeptide. Such glycopeptides can be sialylated using the claimedmethods without prior modification of the glycosylation pattern of theglycopeptide. Alternatively, the methods of the invention can be used tosialylate a peptide that does not include a suitable acceptor; one firstmodifies the peptide to include an acceptor by methods known to those ofskill in the art. In an exemplary embodiment, a GalNAc residue is addedby the action of a GalNAc transferase.

In an exemplary embodiment, the galactosyl acceptor is assembled byattaching a galactose residue to an appropriate acceptor linked to thepeptide, e.g., a GlcNAc. The method includes incubating the peptide tobe modified with a reaction mixture that contains a suitable amount of agalactosyltransferase (e.g., Galβ1,3 or Galβ1,4), and a suitablegalactosyl donor (e.g., UDP-galactose). The reaction is allowed toproceed substantially to completion or, alternatively, the reaction isterminated when a preselected amount of the galactose residue is added.Other methods of assembling a selected saccharide acceptor will beapparent to those of skill in the art.

In yet another embodiment, glycopeptide-linked oligosaccharides arefirst “trimmed,” either in whole or in part, to expose either anacceptor for the sialyltransferase or a moiety to which one or moreappropriate residues can be added to obtain a suitable acceptor. Enzymessuch as glycosyltransferases and endoglycosidases (see, for example U.S.Pat. No. 5,716,812) are useful for the attaching and trimming reactions.In another embodiment of this method, the sialic acid moieties of thepeptide are essentially completely removed (e.g., at least 90, at least95 or at least 99%), exposing an acceptor for a modified sialic acid.

In the discussion that follows, the method of the invention isexemplified by the use of modified sugars having a PEG moiety attachedthereto. The focus of the discussion is for clarity of illustration.Those of skill will appreciate that the discussion is equally relevantto those embodiments in which the modified sugar bears a therapeuticmoiety, biomolecule or the like.

In an exemplary embodiment of the invention in which a carbohydrateresidue is “trimmed” prior to the addition of the modified sugar highmannose is trimmed back to the first generation biantennary structure. Amodified sugar bearing a PEG moiety is conjugated to one or more of thesugar residues exposed by the “trimming back.” In one example, a PEGmoiety is added via a GlcNAc moiety conjugated to the PEG moiety. Themodified GlcNAc is attached to one or both of the terminal mannoseresidues of the biantennary structure. Alternatively, an unmodifiedGlcNAc can be added to one or both of the termini of the branchedspecies.

In another exemplary embodiment, a PEG moiety is added to one or both ofthe terminal mannose residues of the biantennary structure via amodified sugar having a galactose residue, which is conjugated to aGlcNAc residue added onto the terminal mannose residues. Alternatively,an unmodified Gal can be added to one or both terminal GlcNAc residues.

In yet a further example, a PEG moiety is added onto a Gal residue usinga modified sialic acid such as those discussed above.

In another exemplary embodiment, a high mannose structure is “trimmedback” to the mannose from which the biantennary structure branches. Inone example, a PEG moiety is added via a GlcNAc modified with thepolymer. Alternatively, an unmodified GlcNAc is added to the mannose,followed by a Gal with an attached PEG moiety. In yet anotherembodiment, unmodified GlcNAc and Gal residues are sequentially added tothe mannose, followed by a sialic acid moiety modified with a PEGmoiety.

A high mannose structure can also be trimmed back to the elementarytri-mannosyl core.

In a further exemplary embodiment, high mannose is “trimmed back” to theGlcNAc to which the first mannose is attached. The GlcNAc is conjugatedto a Gal residue bearing a PEG moiety. Alternatively, an unmodified Galis added to the GlcNAc, followed by the addition of a sialic acidmodified with a water-soluble sugar. In yet a further example, theterminal GlcNAc is conjugated with Gal and the GlcNAc is subsequentlyfucosylated with a modified fucose bearing a PEG moiety.

High mannose may also be trimmed back to the first GlcNAc attached tothe Asn of the peptide. In one example, the GlcNAc of theGlcNAc-(Fuc)_(a) residue is conjugated with ha GlcNAc bearing a watersoluble polymer. In another example, the GlcNAc of the GlcNAc-(Fuc)aresidue is modified with Gal, which bears a water soluble polymer. In astill further embodiment, the GlcNAc is modified with Gal, followed byconjugation to the Gal of a sialic acid modified with a PEG moiety.

Other exemplary embodiments are set forth in commonly owned U.S. Patentapplication Publications: 20040132640; 20040063911; 20040137557; U.S.patent application Ser. Nos. 10/369,979; 10/410,913; 10/360,770;10/410,945 and PCT/US02/32263 each of which is incorporated herein byreference.

The Examples set forth above provide an illustration of the power of themethods set forth herein. Using the methods described herein, it ispossible to “trim back” and build up a carbohydrate residue ofsubstantially any desired structure. The modified sugar can be added tothe termini of the carbohydrate moiety as set forth above, or it can beintermediate between the peptide core and the terminus of thecarbohydrate.

In an exemplary embodiment, an existing sialic acid is removed from aglycopeptide using a sialidase, thereby unmasking all or most of theunderlying galactosyl residues. Alternatively, a peptide or glycopeptideis labeled with galactose residues, or an oligosaccharide residue thatterminates in a galactose unit. Following the exposure of or addition ofthe galactose residues, an appropriate sialyltransferase is used to adda modified sialic acid.

In another exemplary embodiment, an enzyme that transfers sialic acidonto sialic acid is utilized. This method can be practiced withouttreating a sialylated glycan with a sialidase to expose glycan residuesbeneath the sialic acid. An exemplary polymer-modified sialic acid is asialic acid modified with poly(ethylene glycol). Other exemplary enzymesthat add sialic acid and modified sialic acid moieties onto glycans thatinclude a sialic acid residue or exchange an existing sialic acidresidue on a glycan for these species include ST3Gal3, CST-II,ST8Sia-II, ST8Sia-III and ST8Sia-IV.

In yet a further approach, a masked reactive functionality is present onthe sialic acid. The masked reactive group is preferably unaffected bythe conditions used to attach the modified sialic acid to the Factor IX.After the covalent attachment of the modified sialic acid to thepeptide, the mask is removed and the peptide is conjugated with an agentsuch as PEG. The agent is conjugated to the peptide in a specific mannerby its reaction with the unmasked reactive group on the modified sugarresidue.

Any modified sugar can be used with its appropriate glycosyltransferase,depending on the terminal sugars of the oligosaccharide side chains ofthe glycopeptide. As discussed above, the terminal sugar of theglycopeptide required for introduction of the PEGylated structure can beintroduced naturally during expression or it can be produced postexpression using the appropriate glycosidase(s), glycosyltransferase(s)or mix of glycosidase(s) and glycosyltransferase(s).

In a further exemplary embodiment, UDP-galactose-PEG is reacted withβ1,4-galactosyltransferase, thereby transferring the modified galactoseto the appropriate terminal N-acetylglucosamine structure. The terminalGlcNAc residues on the glycopeptide may be produced during expression,as may occur in such expression systems as mammalian, insect, plant orfungus, but also can be produced by treating the glycopeptide with asialidase and/or glycosidase and/or glycosyltransferase, as required.

In another exemplary embodiment, a GlcNAc transferase, such as GNT1-5,is utilized to transfer PEGylated-GlcNAc to a terminal mannose residueon a glycopeptide. In a still further exemplary embodiment, an the N-and/or O-linked glycan structures are enzymatically removed from aglycopeptide to expose an amino acid or a terminal glycosyl residue thatis subsequently conjugated with the modified sugar. For example, anendoglycanase is used to remove the N-linked structures of aglycopeptide to expose a terminal GlcNAc as a GlcNAc-linked-Asn on theglycopeptide. UDP-Gal-PEG and the appropriate galactosyltransferase isused to introduce the PEG-galactose functionality onto the exposedGlcNAc.

In an alternative embodiment, the modified sugar is added directly tothe peptide backbone using a glycosyltransferase known to transfer sugarresidues to the peptide backbone. Exemplary glycosyltransferases usefulin practicing the present invention include, but are not limited to,GalNAc transferases (GalNAc T1-14), GlcNAc transferases,fucosyltransferases, glucosyltransferases, xylosyltransferases,mannosyltransferases and the like. Use of this approach allows thedirect addition of modified sugars onto peptides that lack anycarbohydrates or, alternatively, onto existing glycopeptides. In bothcases, the addition of the modified sugar occurs at specific positionson the peptide backbone as defined by the substrate specificity of theglycosyltransferase and not in a random manner as occurs duringmodification of a protein's peptide backbone using chemical methods. Anarray of agents can be introduced into proteins or glycopeptides thatlack the glycosyltransferase substrate peptide sequence by engineeringthe appropriate amino acid sequence into the polypeptide chain.

In each of the exemplary embodiments set forth above, one or moreadditional chemical or enzymatic modification steps can be utilizedfollowing the conjugation of the modified sugar to the peptide. In anexemplary embodiment, an enzyme (e.g., fucosyltransferase) is used toappend a glycosyl unit (e.g., fucose) onto the terminal modified sugarattached to the peptide. In another example, an enzymatic reaction isutilized to “cap” sites to which the modified sugar failed to conjugate.Alternatively, a chemical reaction is utilized to alter the structure ofthe conjugated modified sugar. For example, the conjugated modifiedsugar is reacted with agents that stabilize or destabilize its linkagewith the peptide component to which the modified sugar is attached. Inanother example, a component of the modified sugar is deprotectedfollowing its conjugation to the peptide. One of skill will appreciatethat there is an array of enzymatic and chemical procedures that areuseful in the methods of the invention at a stage after the modifiedsugar is conjugated to the peptide. Further elaboration of the modifiedsugar-peptide conjugate is within the scope of the invention.

Enzymes and reaction conditions for preparing the conjugates of thepresent invention are discussed in detail in the parent of the instantapplication as well as co-owned published PCT patent applications WO03/031464, WO 04/033651, WO 04/099231.

In a selected embodiment, a Factor IX peptide, expressed in insectcells, is remodeled such that glycans on the remodeled glycopeptideinclude a GlcNAc-Gal glycosyl residue. The addition of GlcNAc and Galcan occur as separate reactions or as a single reaction in a singlevessel. In this example, GlcNAc-transferase I and Gal-transferase I areused. The modified sialyl moiety is added using ST3Gal-III.

In another embodiment, the addition of GlcNAc, Gal and modified Sia canalso occur in a single reaction vessel, using the enzymes set forthabove. Each of the enzymatic remodeling and glycoPEGylation steps arecarried out individually.

When the peptide is expressed in mammalian cells, different methods areof use. In one embodiment, the peptide is conjugated without need forremodeling prior to conjugation by contacting the peptide with asialyltransferase that transfers the modified sialic acid directly ontoa sialic acid on the peptide forming Sia-Sia-L-R¹, or exchanges a sialicacid on the peptide for the modified sialic acid, forming Sia-L-R¹. Anexemplary enzyme of use in this method is CST-II. Other enzymes that addsialic acid to sialic acid are known to those of skill in the art andexamples of such enzymes are set forth the figures appended hereto.

In yet another method of preparing the conjugates of the invention, thepeptide expressed in a mammalian system is desialylated using asialidase. The exposed Gal residue is sialylated with a modified sialicacid using a sialyltransferase specific for O-linked glycans, providingan Factor IX peptide with an O-linked modified glycan. The desialylated,modified Factor IX peptide is optionally partially or fullyre-sialylated by using a sialyltransferase such as ST3GalIII. UsingST3GalIII with the desialylated peptide, both O and N-linked sites areglycoPEGylated. Using ST3GalI, one or more of the O-linked sites (e.g.,Thr159, Thr169, Thr172 of FIG. 1) are essentially selectivelyglycoPEGylated. The use of CST-II provides a route to essentiallyselectively glycoPEGylate one or more N-linked site (e.g., Asn 157, Asn167 of FIG. 1).

In an exemplary embodiment, the Factor IX of the invention includes atleast one linear 2 kDa PEG moiety covalently attached thereto through anintact glycosyl linking group. A presently preferred Factor IX peptideconjugate of the invention includes up to 9 2 kDa PEG moieties attachedto both N- and O-linked sites through an intact glycosyl linking group,more preferably from 5-9 PEG moieties. In another exemplary embodiment,the Factor IX of the invention includes at least one linear 30 kDa PEGmoiety covalently attached thereto through an intact glycosyl linkinggroup. In a presently preferred embodiment, the Factor IX peptideconjugate of the invention includes from 1 to 3 PEG moieties.

In another aspect, the invention provides a method of making a PEGylatedFactor IX of the invention. The method includes: (a) contacting asubstrate Factor IX peptide comprising a glycosyl group selected from:

with a PEG-sialic acid donor having the formula:

and an enzyme that transfers PEG-sialic acid from said donor onto amember selected from the GalNAc, Gal and the Sia of said glycosyl group,under conditions appropriate for said transfer. An exemplary modifiedsialic acid donor is CMP-sialic acid modified, through a linker moiety,with a polymer, e.g., a straight chain or branched poly(ethylene glycol)moiety. As discussed herein, the peptide is optionally glycosylated withGalNAc and/or Gal and/or Sia (“Remodeled”) prior to attaching themodified sugar. The remodeling steps can occur in sequence in the samevessel without purification of the glycosylated peptide between steps.Alternatively, following one or more remodeling step, the glycosylatedpeptide can be purified prior to submitting it to the next glycosylationor glycPEGylation step.

As illustrated in the examples and discussed further below, placement ofan acceptor moiety for the PEG-sugar is accomplished in any desirednumber of steps. For example, in one embodiment, the addition of GalNActo the peptide can be followed by a second step in which the PEG-sugaris conjugated to the GalNAc in the same reaction vessel. Alternatively,these two steps can be carried out in a single vessel approximatelysimultaneously.

In an exemplary embodiment, the PEG-sialic acid donor has the formula:

In another exemplary embodiment, the PEG-sialic acid donor has theformula:

In a further exemplary embodiment, the Factor IX peptide is expressed inan appropriate expression system prior to being glycopegylated orremodeled. Exemplary expression systems include Sf-9/baculovirus andChinese Hamster Ovary (CHO) cells.

Purification of Factor IX Conjugates

The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the productand one or more of the intermediates, e.g., nucleotide sugars, branchedand linear PEG species, modified sugars and modified nucleotide sugars.Standard, well-known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, column chromatography, ionexchange chromatography, or membrane filtration can be used. It ispreferred to use membrane filtration, more preferably utilizing areverse osmotic membrane, or one or more column chromatographictechniques for the recovery as is discussed hereinafter and in theliterature cited herein. For instance, membrane filtration wherein themembranes have molecular weight cutoff of about 3000 to about 10,000 canbe used to remove proteins such as glycosyl transferases.

If the peptide is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed fragments, is removed.Following glycoPEGylation, the PEGylated peptide is purified byart-recognized methods, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the polypeptide variant from other impurities by one or moresteps selected from immunoaffinity chromatography, ion-exchange columnfractionation (e.g., on diethylaminoethyl (DEAE) or matrices containingcarboxymethyl or sulfopropyl groups), chromatography on Blue-Sepharose,CM Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose,WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl, PhenylToyopearl, or protein A Sepharose, SDS-PAGE chromatography, silicachromatography, chromatofocusing, reverse phase HPLC (e.g., silica gelwith appended aliphatic groups), gel filtration using, e.g., Sephadexmolecular sieve or size-exclusion chromatography, chromatography oncolumns that selectively bind the polypeptide, and ethanol or ammoniumsulfate precipitation.

Modified glycopeptides produced in culture are usually isolated byinitial extraction from cells, enzymes, etc., followed by one or moreconcentration, salting-out, aqueous ion-exchange, or size-exclusionchromatography steps. Additionally, the modified glycoprotein may bepurified by affinity chromatography. Finally, HPLC may be employed forfinal purification steps.

A protease inhibitor, e.g., methylsulfonylfluoride (PMSF) may beincluded in any of the foregoing steps to inhibit proteolysis andantibiotics or preservatives may be included to prevent the growth ofadventitious contaminants.

Within another embodiment, supernatants from systems which produce themodified glycopeptide of the invention are first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a ligand for the peptide, a lectin or antibody molecule boundto a suitable support. Alternatively, an anion-exchange resin may beemployed, for example, a matrix or substrate having pendant DEAE groups.Suitable matrices include acrylamide, agarose, dextran, cellulose, orother types commonly employed in protein purification. Alternatively, acation-exchange step may be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are particularly preferred.

Other methods of use in purification include size exclusionchromatography (SEC), hydroxyapatite chromatography, hydrophobicinteraction chromatography and chromatography on Blue Sepharose. Theseand other useful methods are illustrated in co-assigned U.S. ProvisionalPatent No. (Attorney Docket No. 40853-01-5168-PI, filed May 6, 2005).

One or more RP-HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, may beemployed to further purify a polypeptide conjugate composition. Some orall of the foregoing purification steps, in various combinations, canalso be employed to provide a homogeneous or essentially homogeneousmodified glycoprotein.

The modified glycopeptide of the invention resulting from a large-scalefermentation may be purified by methods analogous to those disclosed byUrdal et al., J. Chromatog. 296: 171 (1984). This reference describestwo sequential, RP-HPLC steps for purification of recombinant human IL-2on a preparative HPLC column. Alternatively, techniques such as affinitychromatography may be utilized to purify the modified glycoprotein.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical composition.The pharmaceutical composition includes a pharmaceutically acceptablediluent and a covalent conjugate between a non-naturally-occurring, PEGmoiety, therapeutic moiety or biomolecule and a glycosylated ornon-glycosylated peptide. The polymer, therapeutic moiety or biomoleculeis conjugated to the peptide via an intact glycosyl linking groupinterposed between and covalently linked to both the peptide and thepolymer, therapeutic moiety or biomolecule.

Pharmaceutical compositions of the invention are suitable for use in avariety of drug delivery systems. Suitable formulations for use in thepresent invention are found in Remington's Pharmaceutical Sciences, MacePublishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

The pharmaceutical compositions may be formulated for any appropriatemanner of administration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous orintramuscular administration. For parenteral administration, such assubcutaneous injection, the carrier preferably comprises water, saline,alcohol, a fat, a wax or a buffer. For oral administration, any of theabove carriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactate polyglycolate) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109.

Commonly, the pharmaceutical compositions are administered parenterally,e.g., intravenously. Thus, the invention provides compositions forparenteral administration that include the compound dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier, e.g.,water, buffered water, saline, PBS and the like. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably from 5 to 9 and most preferably from 7 and 8.

In some embodiments the glycopeptides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, such as phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid-derivatized glycopeptides of the invention.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moieties are available for interaction with the target, forexample, a cell surface receptor. The carbohydrates of the invention maybe attached to a lipid molecule before the liposome is formed usingmethods known to those of skill in the art (e.g., alkylation oracylation of a hydroxyl group present on the carbohydrate with a longchain alkyl halide or with a fatty acid, respectively). Alternatively,the liposome may be fashioned in such a way that a connector portion isfirst incorporated into the membrane at the time of forming themembrane. The connector portion must have a lipophilic portion, which isfirmly embedded and anchored in the membrane. It must also have areactive portion, which is chemically available on the aqueous surfaceof the liposome. The reactive portion is selected so that it will bechemically suitable to form a stable chemical bond with the targetingagent or carbohydrate, which is added later. In some cases it ispossible to attach the target agent to the connector molecule directly,but in most instances it is more suitable to use a third molecule to actas a chemical bridge, thus linking the connector molecule which is inthe membrane with the target agent or carbohydrate which is extended,three dimensionally, off of the vesicle surface.

The compounds prepared by the methods of the invention may also find useas diagnostic reagents. For example, labeled compounds can be used tolocate areas of inflammation or tumor metastasis in a patient suspectedof having an inflammation. For this use, the compounds can be labeledwith ¹²⁵I, ¹⁴C, or tritium.

The active ingredient used in the pharmaceutical compositions of thepresent invention is glycopegylated Factor IX and its derivatives havingthe biological properties of participating in the blood coagulationcascade. The formulation of the present invention is useful as aparenteral formulation in treating coagulation disorders characterizedby low or defective coagulation such as various forms of hemophilia.Preferably, the Factor IX composition of the present invention isadministered parenterally (e.g. IV, IM, SC or IP). Effective dosages areexpected to vary considerably depending on the condition being treatedand the route of administration but are expected to be in the range ofabout 0.1 to 1000 μg/kg body weight of the active material. Preferabledoses for treatment of coagulation disorders are about 50 to about 3000μg/kg three times a week. More preferably, about 500 to about 2000 μg/kgthree times a week. More preferrably, about 750 to about 1500 μg/kgthree times a week, and more preferrably about 1000 μg/kg three times aweek. Because the present invention provides a Factor IX with anenhanced in vivo residence time, the stated dosages are optionallylowered when a composition of the invention is administered.

The following examples are provided to illustrate the conjugates, andmethods and of the present invention, but not to limit the claimedinvention.

Preparative methods for species of use in preparing the compositions ofthe invention are generally set forth in various patent publications,e.g., US 20040137557; WO 04/083258; and WO 04/033651. The followingexamples are provided to illustrate the conjugates, and methods and ofthe present invention, but not to limit the claimed invention.

The following examples are provided to illustrate the conjugates, andmethods and of the present invention, but not to limit the claimedinvention.

EXAMPLES Example 1 Preparation of Cysteine-PEG₂ (2)

1.1 Synthesis of (1)

Potassium hydroxide (84.2 mg, 1.5 mmol, as a powder) was added to asolution of L-cysteine (93.7 mg, 0.75 mmol) in anhydrous methanol (20mL) under argon. The mixture was stirred at room temperature for 30 min,and then mPEG-O-tosylate of molecular mass 20 kilodalton (Ts; 1.0 g,0.05 mmol) was added in several portions over 2 hours. The mixture wasstirred at room temperature for 5 days, and concentrated by rotaryevaporation. The residue was diluted with water (30 mL), and stirred atroom temperature for 2 hours to destroy any excess 20 kilodaltonmPEG-O-tosylate. The solution was then neutralized with acetic acid, thepH adjusted to pH 5.0 and loaded onto a reverse phase chromatography(C-18 silica) column. The column was eluted with a gradient ofmethanol/water (the product elutes at about 70% methanol), productelution monitored by evaporative light scattering, and the appropriatefractions collected and diluted with water (500 mL). This solution waschromatographed (ion exchange, XK 50 Q, BIG Beads, 300 mL, hydroxideform; gradient of water to water/acetic acid-0.75N) and the pH of theappropriate fractions lowered to 6.0 with acetic acid. This solution wasthen captured on a reversed phase column (C-18 silica) and eluted with agradient of methanol/water as described above. The product fractionswere pooled, concentrated, redissolved in water and freeze-dried toafford 453 mg (44%) of a white solid (1). Structural data for thecompound were as follows: ¹H-NMR (500 MHz; D₂O) δ 2.83 (t, 2H, O—C—CH₂—S), 3.05 (q, 1H, S—CHH—CHN), 3.18 (q, 1H, (q, 1H, S—CHH—CHN), 3.38 (s,3H, CH ₃O), 3.7 (t, OCH ₂CH ₂O), 3.95 (q, 1H, CHN). The purity of theproduct was confirmed by SDS PAGE.

1.2 Synthesis of (2)

Triethylamine (˜0.5 mL) was added dropwise to a solution of 1 (440 mg,22 μmol) dissolved in anhydrous CH₂Cl₂ (30 mL) until the solution wasbasic. A solution of 20 kilodalton mPEG-O-p-nitrophenyl carbonate (660mg, 33 μmol) and N-hydroxysuccinimide (3.6 mg, 30.8 μmol) in CH₂Cl₂ (20mL) was added in several portions over 1 h at room temperature. Thereaction mixture was stirred at room temperature for 24 h. The solventwas then removed by rotary evaporation, the residue was dissolved inwater (100 mL), and the pH adjusted to 9.5 with 1.0 N NaOH. The basicsolution was stirred at room temperature for 2 h and was thenneutralized with acetic acid to a pH 7.0. The solution was then loadedonto a reversed phase chromatography (C-18 silica) column. The columnwas eluted with a gradient of methanol/water (the product elutes atabout 70% methanol), product elution monitored by evaporative lightscattering, and the appropriate fractions collected and diluted withwater (500 mL). This solution was chromatographed (ion exchange, XK 50Q, BIG Beads, 300 mL, hydroxide form; gradient of water to water/aceticacid-0.75N) and the pH of the appropriate fractions lowered to 6.0 withacetic acid. This solution was then captured on a reversed phase column(C-18 silica) and eluted with a gradient of methanol/water as describedabove. The product fractions were pooled, concentrated, redissolved inwater and freeze-dried to afford 575 mg (70%) of a white solid (2).Structural data for the compound were as follows: ¹H-NMR (500 MHz; D₂O)δ 2.83 (t, 2H, O—C—CH ₂—S), 2.95 (t, 2H, O—C—CH ₂—S), 3.12 (q, 1H,S—CHH—CHN), 3.39 (s, 3H CH ₃O), 3.71 (t, OCH ₂CH ₂O). The purity of theproduct was confirmed by SDS PAGE.

Example 2 GlycoPEGylation of Factor IX Produced in CHO Cells

This example sets forth the preparation of asialoFactor IX and itssialylation with CMP-sialic acid-PEG.

2. 1 Desialylation of rFactor IX

A recombinant form of Coagulation Factor IX (rFactor IX) was made in CHOcells. 6000 IU of rFactor IX were dissolved in a total of 12 mL USP H₂O.This solution was transferred to a Centricon Plus 20, PL-10 centrifugalfilter with another 6 mL USP H₂O. The solution was concentrated to 2 mLand then diluted with 15 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 5 mMCaCl₂, 0.05% NaN₃ and then reconcentrated. The dilution/concentrationwas repeated 4 times to effectively change the buffer to a final volumeof 3.0 mL. Of this solution, 2.9 mL (about 29 mg of rFactor IX) wastransferred to a small plastic tube and to it was added 530 mUα2-3,6,8-Neuraminidase-agarose conjugate (Vibrio cholerae, Calbiochem,450 μL). The reaction mixture was rotated gently for 26.5 hours at 32°C. The mixture was centrifuged 2 minutes at 10,000 rpm and thesupernatant was collected. The agarose beads (containing neuraminidase)were washed 6 times with 0.5 mL 50 mM Tris-HCl pH 7.12, 1 M NaCl, 0.05%NaN₃. The pooled washings and supernatants were centrifuged again for 2minutes at 10,000 rpm to remove any residual agarose resin. The pooled,desialylated protein solution was diluted to 19 mL with the same bufferand concentrated down to ˜2 mL in a Centricon Plus 20 PL-10 centrifugalfilter. The solution was twice diluted with 15 mL of 50 mM Tris-HCl pH7.4, 0.15 M NaCl, 0.05% NaN₃ and reconcentrated to 2 mL. The finaldesialyated rFactor IX solution was diluted to 3 mL final volume (˜10mg/mL) with the Tris Buffer. Native and desialylated rFactor IX sampleswere analyzed by IEF-Electrophoresis. Isoelectric Focusing Gels (pH 3-7)were run using 1.5 μL (15 μg) samples first diluted with 10 μL Trisbuffer and mixed with 12 μL sample loading buffer. Gels were loaded, runand fixed using standard procedures. Gels were stained with ColloidalBlue Stain (FIG. 154), showing a band for desialylated Factor IX.

Example 3 Preparation of PEG (1 kDa and 10 kDa)-SA-Factor IX

Desialylated rFactor-IX (29 mg, 3 mL) was divided into two 1.5 mL (14.5mg) samples in two 15 mL centrifuge tubes. Each solution was dilutedwith 12.67 mL 50 mM Tris-HCl pH 7.4, 0.15 M NaCl, 0.05% NaN₃ and eitherCMP-SA-PEG-1k or 10k (7.25 μmol) was added. The tubes were invertedgently to mix and 2.9 U ST3Gal3 (326 μL) was added (total volume 14.5mL). The tubes were inverted again and rotated gently for 65 hours at32° C. The reactions were stopped by freezing at −20° C. 10 μg samplesof the reactions were analyzed by SDS-PAGE. The PEGylated proteins werepurified on a Toso Haas Biosep G3000SW (21.5×30 cm, 13 um) HPLC columnwith Dulbecco's Phosphate Buffered Saline, pH 7.1 (Gibco), 6 mL/min. Thereaction and purification were monitored using SDS Page and IEF gels.Novex Tris-Glycine 4-20% 1 mm gels were loaded with 10 μL (10 μg) ofsamples after dilution with 2 μL of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl,0.05% NaN₃ buffer and mixing with 12 μL sample loading buffer and 1 μL0.5 M DTT and heated for 6 minutes at 85° C. Gels were stained withColloidal Blue Stain (FIG. 155) showing a band for PEG (1 kDa and 10kDa)-SA-Factor IX.

Example 4 Direct Sialyl-GlycoPEGylation of Factor IX

This example sets forth the preparation of sialyl-PEGylation of FactorIX without prior sialidase treatment.

4.1 Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(10 KDa)

Factor IX (1100 IU), which was expressed in CHO cells and was fullysialylated, was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2%sucrose, 0.05% NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(10kDa) (27 mg, 2.5 μmol) was then dissolved in the solution and 1 U ofST3Gal3 was added. The reaction was complete after gently mixing for 28hours at 32° C. The reaction was analyzed by SDS-PAGE as described byInvitrogen. The product protein was purified on an Amersham Superdex 200(10×300 mm, 13 μm) HPLC column with phosphate buffered saline, pH 7.0(PBS), 1 mL/min. R_(t)=9.5 min.

Example 5 Sialyl-PEGylation of Factor-IX with CMP-SA-PEG-(20 kDa)

Factor IX (1100 IU), which was expressed in CHO cells and was fullysialylated, was dissolved in 5 mL of 20 mM histidine, 520 mM glycine, 2%sucrose, 0.05% NaN₃ and 0.01% polysorbate 80, pH 5.0. The CMP-SA-PEG-(20kDa) (50 mg, 2.3 μmol) was then dissolved in the solution and CST-II wasadded. The reaction mixture was complete after gently mixing for 42hours at 32° C. The reaction was analyzed by SDS-PAGE as described byInvitrogen.

The product protein was purified on an Amersham Superdex 200 (10×300 mm,13 μm) HPLC column with phosphate buffered saline, pH 7.0 (Fisher), 1mL/min. R_(t)=8.6 min.

Example 6 Sialic Acid Capping of GlycoPEGylated Factor IX

This examples sets forth the procedure for sialic acid capping ofsialyl-glycoPEGylated peptides. Here, Factor-IX is the exemplarypeptide.

6.1 Sialic Acid Capping of N-Linked and O-Linked Glycans ofFactor-IX-SA-PEG (10 kDa)

Purified r-Factor-IX-PEG (10 kDa) (2.4 mg) was concentrated in aCentricon® Plus 20 PL-10 (Millipore Corp., Bedford, Mass.) centrifugalfilter and the buffer was changed to 50 mM Tris-HCl pH 7.2, 0.15 M NaCl,0.05% NaN₃ to a final volume of 1.85 mL. The protein solution wasdiluted with 372 μL of the same Tris buffer and 7.4 mg CMP-SA (12 μmol)was added as a solid. The solution was inverted gently to mix and 0.1 UST3Gall and 0.1 U ST3Gal3 were added. The reaction mixture was rotatedgently for 42 hours at 32° C.

A 10 μg sample of the reaction was analyzed by SDS-PAGE. NovexTris-Glycine 4-12% 1 mm gels were performed and stained using ColloidalBlue as described by Invitrogen. Briefly, samples, 10 μL (10 μg), weremixed with 12 μL sample loading buffer and 1 μL 0.5 M DTT and heated for6 minutes at 85° C. (FIG. 156, lane 4).

Example 7 Glycopegylated Factor IX Pharmacokinetic Study

Four glycoPEGylated FIX variants (PEG-9 variants) were tested in a PKstudy in normal mice. The activity of the four compounds had previouslybeen established in vitro by clot, endogenous thrombin potential (ETP),and thromboelastograph (TEG) assays. The activity results are summarizedin Table I.

ETP TEG Clot activity (relative (relative Compound (% of plasma)specific activity specific activity BeneFIX 45% 1.0 1.0 PEG-9-2K (LS)27% 0.3 0.2 PEG-9-2K (HS) 20% 0.2 0.1 PEG-9-10K 11% 0.6 0.3 PEG-9-30K14% 0.9 0.4

To assess the prolongation of activity of the four PEG-9 compounds incirculation, a PK study was designed and performed. Non-hemophilic micewere used, 2 animal per time point, 3 samples per animal. Sampling timepoints were 0, 0.08, 0.17, 0.33, 1, 3, 5, 8, 16, 24, 30, 48, 64, 72, and96 h post compound administration. Blood samples were centrifuged andstored in two aliquots; one for clot analysis and one for ELISA. Due tomaterial restrictions, the PEG-9 compounds were dosed in differentamounts: BeneFIX 250 U/kg; 2K(low substitution: “LS” (1-2 PEGsubstitutions per peptide molecule) 200 U/kg; 2K(high substitution: “HS”(3-4 PEG substitutions per peptide molecule) 200 U/kg; 10K 100 U/kg; 30K100 U/kg. All doses were based on measured clotting assay units.

The results are outlined in FIG. 6 and Table II.

TABLE II Dose Cmax AUC CL Compound (U/kg) (U/mL) (h-U/mL (mL/h/kg)BeneFIX 250 0.745 1.34 187 PEG-9-2K (LS) 200 0.953 4.69 42.7 PEG-9-2K(HS) 200 0.960 9.05 22.1 PEG-9-10K 100 0.350 2.80 35.7 PEG-9-30K 1001.40 8.83 11.3

The results demonstrate a trend towards prolongation for all the PEG-9compounds. The values of AUC and Cmax were not compared directly.However, clearance (CL) was compared and CL is lower for the PEG-9compounds compared to BeneFIX, indicating a longer residence time in themice. The time for the last detectable clot activity is increased forthe PEG-9 compounds compared to BeneFIX, even though BeneFIX wasadministered at the highest dose.

Example 8 Preparation of Ls and Hs Glycopegylated Factor IX

GlycoPEGylated Factor IX with a low degree of substitution with PEG wereprepared from native Factor IX by an exchange reaction catalyzed byST3Gal-III. The reactions were performed in a buffer of 10 mM histidine,260 mM glycine, 1% sucrose and 0.02% Tween 80, pH 7.2. For PEGylationwith CMPSA-PEG (2 kD and 10 kD), Factor IX (0.5 mg/mL) was incubatedwith ST3GalIII (50 mU/mL) and CMP-SA-PEG (0.5 mM) for 16 h at 32° C. ForPEGylation with CMP-SA-PEG 30 kD, the concentration of Factor IX wasincreased to 1.0 mg/mL, and the concentration of CMP-SA-PEG wasdecreased to 0.17 mM. Under these conditions, more than 90% of theFactor IX molecules were substituted with at least one PEG moiety.

GlycoPEGylated Factor IX with a high degree of substitution with PEGwere prepared by enzymatic desialylation of native Factor IX. The FactorIX peptide was buffer exchanged into 50 mM mES, pH 6.0, using a PD10column, adjusted to a concentration of 0.66 mg/mL and treated with AUSsialidase (5 mU/mL) for 16 h at 32° C. Desialylation was verified bySDS-PAGE, HPLC and MALDI glycan analysis. Asialo Factor IX was purifiedon Q Sepharose FF to remove the sialidase. The CaCl₂ fraction wasconcentrated using an Ultra15 concentrator and buffer exchanged intoMES, pH 6.0 using a PD10 column.

2 kD and 10 kD PEGylation of asialo-Factor IX (0.5 mg/mL) was carriedout by incubation with ST3Gal-III (50 mU/mL) and CMP-SA-PEG (0.5 mM) at32° C. for 16 h. For PEGylation with CMPSA-PEG-30 kD, the concentrationof Factor IX was increased to 1.0 mg/mL and the concentration ofCMP-SA-PEG was decreased to 0.17 mM. After 16 h of PEGylation, glycanswith terminal galactose were capped with sialic acid by adding 1 mMCMP-SA and continuing the incubation for an additional 8 h at 32° C.Under these conditions, more than 90% of the Factor IX molecules weresubstituted with at least one PEG moiety. Factor IX produced by thismethod has a higher apparent molecular weight in SDS-PAGE.

Example 9 Preparation of O-Glycopegylated Factor IX

O-glycan chains were introduced de novo into native Factor IX (1 mg/mL)by incubation of the peptide with GalNAcT-II (25 mU/mL) and 1 mMUDP-GalNAc at 32° C. After 4 h of incubation, the PEGylation reactionwas initiated by adding CMPSA-PEG (2 Kd or 10 Kd at 0.5 mM or 30 kDd at0.17 mM) and ST6GalNAc-I (25 mU/mL) and incubating for an additional 20h.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A Factor IX peptide conjugate comprising a glycosyl linking groupattached to an amino acid residue of said peptide, said glycosyl linkinggroup comprising a modified sialyl residue having the formula:

wherein R² is H, CH₂OR⁷, COOR⁷ or OR⁷ wherein R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R³ and R⁴ are members independently selected from H,substituted or unsubstituted alkyl, OR⁸, NHC(O)R⁹ wherein R⁸ and R⁹ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl or sialic acid; L^(a) is alinker selected from a bond, substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl R¹⁶ and R¹⁷ are independentlyselected polymeric arms; X² and X⁴ are independently selected linkagefragments joining polymeric moieties R¹⁶ and R¹⁷ to C; and X⁵ is anon-reactive group.
 2. The peptide conjugate according to claim 1,wherein the moiety:

has a formula that is a member selected from:

wherein Q is selected from H and substituted or unsubstituted C₁-C₆alkyl; e and f are integers independently selected from 1 to 2500; and qis an integer from 0 to
 20. 3. The peptide conjugate according to claim2, wherein said moiety has a formula that is a member selected from:

wherein Q is selected from H and substituted or unsubstituted C₁-C₆alkyl; e, f and f′ are integers independently selected from 1 to 2500;and q and q′ are integers independently selected from 1 to
 20. 4. Thepeptide conjugate according to claim 1, wherein said glycosyl linkinggroup has a formula selected from:


5. The peptide conjugate according to claim 4, wherein said glycosyllinking group has the formula:


6. The peptide conjugate according to claim 5, wherein said glycosyllinking group attached to said amino acid residue has the formula:

wherein AA is said amino acid residue of said peptide.
 7. The peptideconjugate according to claim 6, wherein said amino acid residue is amember selected from serine or threonine.
 8. The peptide conjugateaccording to claim 1, wherein said peptide has the amino acid sequenceof SEQ. ID. NO:
 1. 9. The peptide according to claim 8, wherein saidamino acid residue is a serine at position 61 of SEQ. ID. NO:1.
 10. Thepeptide conjugate according to claim 1, wherein said peptide comprisesat least one glycosyl linking group comprising a substructure having theformula:

wherein R¹⁵ is said modified sialyl residue; and p is an integer from 1to
 10. 11. The peptide conjugate according to claim 10, wherein said atleast one glycosyl linking group attached to an amino acid of saidpeptide has a formula selected from:

and combinations thereof wherein AA is said amino acid residue of saidpeptide; t is an integer equal to 0 or 1; p is an integer from 1 to 10;and R^(15′) is a member selected from H, OH, sialic acid, said modifiedsialyl residue and Sia-Sia^(p) wherein Sia^(p) is said modified sialylresidue, wherein at least one R^(15′) is selected from said modifiedsialyl residue and Sia-Sia^(p).
 12. The peptide conjugate according toclaim 11, wherein said amino acid residue is an asparagine residue. 13.The peptide conjugate according to claim 12, wherein said peptide hasthe amino acid sequence of SEQ ID NO:
 1. 14. The peptide conjugateaccording to claim 13, wherein said glycosyl residue is attached to amember selected from Asn 157, Asn 167 and combinations thereof.
 15. Amethod of preparing a peptide conjugate according to claim 1, saidmethod comprising: (a) contacting a substrate Factor IX peptidecomprising a glycosyl moiety selected from:

with a PEG-sialic acid donor having the formula:

wherein c is 0 or 1; and (b) contacting said Factor IX and saidPEG-sialic acid donor with an enzyme that transfers PEG-sialic acid fromsaid donor onto said glycosyl moiety, under conditions appropriate forsaid transfer.
 16. The method of claim 15, further comprising, prior tostep (a): (b) expressing said substrate Factor IX peptide in a suitablehost.
 17. The method of claim 16, wherein said host is selected from aninsect cell and a mammalian cell.
 18. The method of claim 17, whereinsaid insect cell is a Spodoptera frugiperda cell line.
 19. A method ofstimulating blood coagulation in a mammal, said method comprisingadministering to said mammal a peptide conjugate according to claim 1 inan amount sufficient to stimulate said blood coagulation.
 20. A methodof treating hemophilia in a subject, said method comprisingadministering to said subject a peptide conjugate according to claim 1in an amount effective to treat said hemophilia.
 21. A pharmaceuticalformulation comprising the Factor IX peptide conjugate according toclaim 1, and a pharmaceutically acceptable carrier.
 22. A Factor IXpeptide conjugate comprising a glycosyl linking group attached to anamino acid residue of said peptide, said glycosyl linking groupcomprising a modified sialyl residue having the formula:

wherein R² is H, CH₂OR⁷, COOR⁷, COO⁻ or OR⁷ wherein R⁷ represents H,substituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl; R³ and R⁴ are members independently selected from H,substituted or unsubstituted alkyl, OR⁸, NHC(O)R⁹ wherein R⁸ and R⁹ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl or sialic acid; s is an integerfrom 1 to 20; f is an integer from 1 to 2500; and Q is a member selectedfrom H and substituted or unsubstituted C₁-C₆ alkyl.
 23. The peptideconjugate according to claim 22, wherein said modified sialyl residuehas the formula:


24. The peptide conjugate according to claim 23, wherein Q is selectedfrom H and CH₃.
 25. The peptide conjugate according to claim 22, whereinsaid peptide comprises at least one glycosyl linking group comprising asubstructure having the formula:

wherein R¹⁵ is said modified sialyl residue; and p is an integer from 1to
 10. 26. The peptide conjugate according to claim 25, wherein said atleast one glycosyl linking group attached to an amino acid of saidpeptide has a formula selected from:

and combinations thereof wherein AA is said amino acid residue of saidpeptide; t is an integer equal to 0 or 1; p is an integer from 1 to 10;and R^(15′) is a member selected from H, OH, sialic acid, said modifiedsialyl residue and Sia-Sia^(p) wherein Sia^(p) is said modified sialylresidue, wherein at least one R^(15′) is selected from said modifiedsialyl residue and Sia-Sia^(p).
 27. The peptide conjugate according toclaim 22, wherein said amino acid residue is asparagine.
 28. The peptideconjugate according to claim 27, wherein said peptide has the amino acidsequence of SEQ ID NO:
 1. 29. The peptide conjugate according to claim28, wherein said glycosyl residue is attached to a member selected fromAsn 157, Asn 167 and combinations thereof.
 30. The peptide conjugateaccording to claim 22, wherein said glycosyl linking group comprises theformula:

in which b is 0 or
 1. 31. The peptide conjugate according to claim 30,wherein s is 1; and f is an integer from about 200 to about
 300. 32. Thepeptide conjugate according to claim 22, wherein said amino acid residueis a member selected from serine or threonine.
 33. The peptide conjugateaccording to claim 32, wherein said peptide has the amino acid sequenceof SEQ. ID. NO:1.
 34. The peptide conjugate according to claim 33,wherein said amino acid residue is a serine at position 61 of SEQ. ID.NO:1.
 35. A method of preparing the peptide conjugate according to claim22, said method comprising: (a) contacting a substrate Factor IX peptidecomprising a glycosyl moiety selected from:

with a PEG-sialic acid donor having the formula:

wherein c is 0 or 1; r is 0 or 1; and (b) contacting Factor IX and saidPEG-sialic acid donor with an enzyme that transfers PEG-sialic acid fromsaid donor onto said glycosyl moiety, under conditions appropriate forsaid transfer.
 36. The method of claim 35, further comprising, prior tostep (a): (b) expressing said substrate Factor IX peptide in a suitablehost.
 37. The method of claim 36, wherein said host an insect cell. 38.The method of claim 37, wherein said insect cell is a Spodopterafrugiperda cell line.
 39. A method of stimulating blood coagulation in amammal, said method comprising administering to said mammal a peptideconjugate according to claim 22 in an amount sufficient to stimulatesaid blood coagulation.
 40. A method of treating hemophilia in asubject, said method comprising administering to said subject a peptideconjugate according to claim 22 in an amount effective to treat saidhemophilia.
 41. A pharmaceutical formulation comprising the Factor IXpeptide conjugate according to claim 22, and a pharmaceuticallyacceptable carrier.